Apparatus and methods for controlling sonic treatment

ABSTRACT

Apparatus and methods are disclosed for treating a sample by selectively controlling sonic energy and/or selectively controlling the location of the sample relative to the sonic energy.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S.provisional application Ser. Nos. 60/105,933, filed Oct. 28, 1998;60/110,460, filed Dec. 1, 1998; 60/119,500, filed Feb. 10, 1999;60/143,440, filed Jul. 13, 1999; and 60/148,279, filed Aug. 11, 1999,and incorporates herein by reference the entirety of each of thedisclosures of these provisional applications.

TECHNICAL FIELD

[0002] The present invention generally relates to the field ofcontrolled sonic energy emitting devices for treating material,particularly biological material.

BACKGROUND OF THE INVENTION

[0003] Ultrasonics have been utilized for many years for a variety ofdiagnostic, therapeutic, and research purposes. The acoustic physics ofultrasonics is well understood; however, the biophysical, chemical, andmechanical effects are generally only empirically understood. Some usesof sonic or acoustic energy in materials processing include“sonication,” an unrefined process of mechanical disruption involvingthe direct immersion of an unfocused ultrasound source emitting energyin the kilohertz (“kHz”) range into a fluid suspension of the materialbeing treated. Accordingly, the sonic energy often does not reach atarget in an effective dose because the energy is scattered, absorbed,and/or not properly aligned with the target. There are also specificclinical examples of the utilization of therapeutic ultrasound (e.g.,lithotripsy) and of diagnostic ultrasound (e.g., fetal imaging).However, ultrasonics have heretofore not been controlled to provide anautomated, broad range, precise materials processing or reaction controlmechanism.

SUMMARY OF THE INVENTION

[0004] The present invention relates to apparatus and methods forselectively exposing a sample to sonic energy, such that the sample isexposed to produce a desired result such as, but without limitation,heating the sample, cooling the sample, fluidizing the sample, mixingthe sample, stirring the sample, disrupting the sample, permeabilizing acomponent of the sample, enhancing a reaction in the sample, andsterilizing the sample. For example, altering the permeability oraccessibility of a material, especially labile biological materials, ina controlled manner can allow for manipulation of the material whilepreserving the viability and/or biological activity of the material. Inanother example, mixing materials or modulating transport of a componentinto or out of materials, in a reproducible, uniform, automated manner,can be beneficial. According to one embodiment of the system, sampleprocessing control includes a feedback loop for regulating at least oneof sonic energy location, pulse pattern, pulse intensity, and absorbeddose of the ultrasound. The system can be automated. In one embodiment,the ultrasonic energy is in the megahertz (MHz) frequency range, incontrast to classical sonic processing which typically employsultrasonic energy in the kilohertz (kHz) frequency range.

[0005] When ultrasonic energy interacts with a complex biological orchemical system, the acoustic field often becomes distorted, reflected,and defocused. The net effect is that energy distribution becomesnon-uniform and/or defocused compared to the input. Non-uniform reactionconditions can limit reaction applications to non-critical processes,such as bulk fluid treatment where temperature gradients within a sampleare inconsequential. However, some of the non-uniform aspects are highlydeleterious to samples, such as extreme temperature gradients thatdamage sample integrity. For example, in some instances, the hightemperature would irreversibly denature target proteins. As aconsequence, many potential applications of ultrasound, especiallybiological applications, are limited to specific, highly specializedapplications, such as lithotripsy and diagnostic imaging, because of thepotentially undesirable and uncontrollable aspects of ultrasound incomplex systems.

[0006] Typically, when ultrasound is applied to a bulk biological samplesolution, such as for the extraction of intracellular constituents fromtissue, the treatment causes a complex, heterogeneous, mixture ofsub-events that vary during the course of a treatment dose. In otherwords, the ultrasonic energy may be partitioned between various states.For example, the energy may directly treat a sample or the energy mayspatially displace a target moiety and shift the target out of theoptimal energy zone. Additionally or alternatively, the energy mayresult in interference that reflects the acoustic energy. For example, a“bubble shield” occurs when a wave front of sonic energy createscavitation bubbles that persist until the next wave front arrives, suchthat the energy of the second wave front is at least partially blockedand/or reflected by the bubbles. Still further, larger particles in thesample may move to low energy nodes, thereby leaving the smallerparticles in the sample with more dwell-time in the high energy nodes.In addition, the sample viscosity, temperature, and uniformity may varyduring the ultrasonic process, resulting in gradients of theseparameters during processing. Accordingly, current processes aregenerally random and non-uniform, especially when applied to in vitroapplications, such as membrane permeabilization, hindering the use ofultrasound in high throughput applications where treatmentstandardization from one sample to the next is required.

[0007] Processing samples containing labile material, in particularbiological material, is still largely a manual process, and poorlyadapted to high-throughput sample processing required for applicationssuch as pharmaceutical and agricultural genomics. For example, exceptfor isolated or exposed cells, the insertion of a nucleic acid into asample, for temporary or permanent transformation, is stillsubstantially manual. Most transformation techniques have been developedfor a small subset of materials, which typically have only a singleplasma membrane separating their interior from the environment. Thesemembranes may be permeabilized using detergents, salts, osmotic shock,or simple freeze-thawing. Thus, materials such as viruses, culturedcells, and bacteria and protists, such as yeast, which have been treatedto prevent the formation of cell walls, can be transfected by any of anumber of standard methods. For example, transfection can be undertakenwith vectors including viruses that bind to plasma membranes for directtransport, and can be undertaken in a direct transfection with “naked”DNA that is often coated with cationic lipids or polymers or that is inthe presence of chemical or biochemical membrane permeabilizing agents.

[0008] Moreover, many biological materials of interest have supportingstructures, and are significantly harder to permeabilize or otherwise toaccess the plasma membrane with macromolecular agents or viruses. Thesupporting structures range from simple cell walls, as in yeast, tocomplex protein and glycoprotein structures, as in animal tissue, totenacious and only slowly degradable polysaccharide structures, as inplants and insects, to physically durable mineralized supports, as indiatoms and bone. In all of these “hard” materials, physical disruptionof the supporting matrices is required typically to precede or accompanytransfection or other nucleic acid insertion to allow reliableintroduction of extracellular components.

[0009] Sonication has been used to break up difficult materials such asplant tissue. Sonication, typically implemented by vibration of a probeat frequencies of 10,000 Hz or higher, creates shearing forces within aliquid sample. However, the resultant shear is not readily controlled,so that when sufficient energy is applied to disrupt a supportingmatrix, the shear will also tend to destroy fragile intracellularstructures. Indeed, sonication is routinely used to randomly shear DNAin solution into small fragments. Such fragmentation limits theusefulness of these techniques for many purposes, and particularly fortransfection, which requires a viable cell to be successful.

[0010] The present invention addresses these problems and providesapparatus and methods for the non-contact treatment of samples withultrasonic energy, using a focused beam of energy. The frequency of thebeam can be variable and can be in the range of about 100 kHz to 100MHz, more preferably 500 kHz to 10 MHz. For example, the presentinvention can treat samples with ultrasonic energy while controlling thetemperature of the sample, by use of computer-generated complexwavetrains, which may further be controlled by the use of feedback froma sensor. The acoustic output signal, or wavetrain, can vary in any orall of frequency, intensity, duty cycle, burst pattern, and pulse shape.In another example, the present invention can treat samples withultrasonic energy when the samples are in an array, and individualsamples in the array may be treated differentially or identically.Moreover, this treatment can be undertaken automatically under computercontrol. In another example, the present invention can treat sampleswith ultrasonic energy in a uniform way over the entire sample, by therelative movement of the sample and the focus of the beam, in any or allof two or three dimensions.

[0011] The apparatus and methods of the present invention can becontrolled by a computer program. In one embodiment, the sequence ofactions taken by the computer is predetermined. Such embodiments can beuseful in high-speed, high-volume processing. In another embodiment, theprocesses are enhanced with a program that uses feedback control tomodify or determine the actions thereof, using techniques includingalgorithmic processing of input, the use of lookup tables, and similarintegration devices and processes.

[0012] A feedback control mechanism, in connection with any of accuracy,reproducibility, speed of processing, control of temperature, provisionof uniformity of exposure to sonic pulses, sensing of degree ofcompletion of processing, monitoring of cavitation, and control of beamproperties (including intensity, frequency, degree of focusing,wavetrain pattern, and position), can enhance certain embodiments of thepresent invention. A variety of sensors or sensed properties may beappropriate for providing input for feedback control. These propertiescan include sensing of temperature of the sample; sonic beam intensity;pressure; bath properties including temperature, salinity, and polarity;sample position; and optical or visual properties of the samples. Theseoptical properties may include apparent color, emission, absorption,fluorescence, phosphorescence, scattering, particle size, laser/Dopplerfluid and particle velocities, and effective viscosity. Sample integrityor comminution can be sensed with a pattern analysis of an opticalsignal. Any sensed property or combination thereof can serve as inputinto a control system. The feedback can be used to control any output ofthe system, for example beam properties, sample position, and treatmentduration.

[0013] The samples can be treated in any convenient vessel or container.Vessels can be sealed for the duration of the treatment to preventcontamination of the sample or of the environment. Arrays of vessels canbe used for processing large numbers of samples. These arrays can bearranged in one or more high throughput configurations. Examples includemicrotiter plates, typically with a temporary sealing layer to close thewells, blister packs, similar to those used to package pharmaceuticalssuch as pills and capsules, and arrays of polymeric bubbles, similar tobubble wrap, preferably with a similar spacing to typical microtiterwells. The latter are described in more detail below.

[0014] The treatment, which may be performed or enhanced by use ofultrasonic wavetrains, include any unit operation which is susceptibleto being implemented or is enhanced by sonic waves or pulses. Inparticular, these results include lysing, extracting, permeabilizing,stirring or mixing, comminuting, heating, fluidizing, sterilizing,catalyzing, and selectively degrading. Sonic waves may also enhancefiltration, fluid flow in conduits, and fluidization of suspensions.Processes of the invention may be synthetic, analytic, or simplyfacilitative of other processes such as stirring.

[0015] Any sample is potentially suitable for processing by thetechniques and apparatuses of the invention. For example, any materialthat includes biological organisms or material derived therefrom issuitable. Many chemicals can be processed more efficiently, particularlyin small-scale or combinatorial reactions or assays, with the processesof the invention, including remote, non-contact mixing or stirring.Physical objects, such as mineral samples and particulates includingsands and clays, also can be treated with the present invention.

[0016] According to the present invention, several aspects of theinvention can enhance the reproducibility and/or effectiveness ofparticular treatments using ultrasonic energy in in vitro applications,where reproducibility, uniformity, and precise control are desired.These aspects include the use of feedback, precise focusing of theultrasonic energy, monitoring and regulating of the acoustic waveform(including frequency, amplitude, duty cycle, and cycles per burst),positioning of the reaction vessel relative to the ultrasonic energy sothat the sample is uniformly treated, controlling movement of the samplerelative to the focus of ultrasonic energy during a processing step,and/or controlling the temperature of the sample being treated, eitherby the ultrasonic energy parameters or through the use of temperaturecontrol devices such as a water bath. A treatment protocol can beoptimized, using one or a combination of the above variables, tomaximize, for example, shearing, extraction, permeabilization,comminution, stirring, or other process steps, while minimizingundesirable thermal effects.

[0017] In one embodiment of the invention, high intensity ultrasonicenergy is focused on a reaction vessel, and “real time” feedbackrelating to one or more process variables is used to control theprocess. In another embodiment, the process is automated and is used ina high throughput system, such as a 96-well plate, or a continuousflowing stream of material to be treated, optionally segmented.

[0018] Minimization of unwanted interference with the pattern of appliedultrasonic energy is another feature of the invention. For example,ultrasonic energy applied to a sample in a reaction vessel has thepotential to directly interact with the target sample, or to reflectfrom bubbles or other effects from a previous cycle of ultrasoundapplication and not interact with the target, or to miss the targetbecause of spatial separation or mismatch. Minimization of interferenceis especially beneficial for remote, automated, sterile processing ofsmall amounts of target material, for example, 10 mg of a biopsy tissue.By minimizing the reflections and optimizing spatial positioning, theultrasonic energy is more efficiently utilized and controlled. Theprocess can be standardized to obtain reproducibility by presettingconditions such as waveform and positioning, by a feedback signal andfeedback-based control to maintain preset performance target parameters,or by a combination of these methods.

[0019] In certain embodiments, the processing system can include a highintensity transducer that produces acoustic energy when driven by anelectrical or optical energy input; a device or system for controllingexcitation of the transducer, such as an arbitrary waveform generator,an RF amplifier, and a matching network for controlling parameters suchas time, intensity, and duty cycle of the ultrasonic energy; apositioning system such as a 2-dimensional (x, y) or a 3-dimensional (x,y, z) positioning system that can be computer controlled to allowautomation and the implementation of feedback from monitoring; atemperature sensor; a device for controlling temperature; one or morereaction vessels; and a sensor for detecting, for example, optical,radiative, and/or acoustic signatures.

[0020] Vessels containing the samples can be sealed during theprocessing, and hence can be sterile throughout, or after, theprocedure. Moreover, the use of focused ultrasound allows the samples inthe vessels to be processed, including processing by stirring, withoutcontacting the samples, even when the vessels are not sealed.

[0021] The processes have a variety of applications, including, butwithout limitation, extraction, permeabilization, mixing, comminuting,sterilization, flow control, and reacting. For example, mixing in avessel can be achieved with temperature fluctuations controlled towithin about plus or minus one degree Celsius. More precise control ispossible, if required. In another example, labile biological materialscan be extracted from plant materials without loss of activity or theuse of harsh solvents. In other applications, complex cells can bepermeabilized and molecules such as nucleotide molecules can beintroduced into the cells using the process of the invention. Otherapplications include modulating binding reactions that are useful inseparations, biological assays, and hybridization reactions.

[0022] One aspect of the invention includes an apparatus for processinga sample using sonic energy. The apparatus includes a sonic energysource for emitting sonic energy; a holder for the sample, the samplemovable relative to the emitted sonic energy; and a processor forcontrolling the sonic energy source and location of the sample accordingto a predetermined methodology, such that the sample is selectivelyexposed to sonic energy to produce a desired result. The desired resultcan be heating the sample, cooling the sample, fluidizing the sample,mixing the sample, stirring the sample, disrupting the sample,increasing permeability of a component of the sample, enhancing areaction within the sample, and/or sterilizing the sample. Also, thedesired result can be an in vitro or an ex vivo treatment.

[0023] This aspect and other aspects of the invention can include any orall of the following features. The apparatus can further include afeedback system connected to the processor for monitoring at least onecondition to which the sample is subjected during processing, such thatthe processor controls at least one of the sonic energy source and thelocation of the sample in response to the at least one condition. Thefeedback system can include a sensor for monitoring the at least onecondition. The apparatus can further include a temperature control unitfor controlling temperature of the sample, and the processor can controlthe temperature control unit. The apparatus can further include apressure control unit for controlling pressure to which the sample isexposed, and the processor controls the pressure control unit. The sonicenergy source can include a transducer. The transducer can focus thesonic energy and can include at least one piezoelectric element, anarray of piezoelectric elements, an electrohydraulic element, amagnetostrictive element, an electromagnetic transducer, a chemicalexplosive element, and/or a laser-activated element. A piezoelectricelement can include a spherical transmitting surface oriented such thatthe focal axis is oriented vertically or in any other predetermineddirection. The holder can support a sample container for containing thesample. The sample container can be a membrane pouch, thermopolymerwell, polymeric pouch, hydrophobic membrane, microtiter plate,microtiter well, test tube, centrifuge tube, microfuge tube, ampoule,capsule, bottle, beaker, flask, and/or capillary tube. The samplecontainer can form multiple compartments and can include a rupturablemembrane for transferring a fraction of the sample away from the holder.The apparatus can further include a device for moving the sample from afirst location to a second location, such as a stepper motor. Theapparatus can also include an acoustically transparent material disposedbetween the sonic energy source and the holder. The sample can flowthrough a conduit. The sonic energy source can generate sonic energy attwo or more different frequencies, optionally in the form of a serialwavetrain. The wavetrain can include a first wave component and adifferent second wave component. Alternatively or additionally, thewavetrain can include about 1000 cycles per burst at about a 10% dutycycle at about a 500 mV amplitude.

[0024] Another aspect of the invention relates to a method forprocessing a sample with sonic energy. The method includes the steps ofexposing the sample to sonic energy and controlling at least one of thesonic energy and location of the sample relative to the sonic energyaccording to a predetermined methodology, such that the sample isselectively exposed to sonic energy to produce a desired result. Thedesired result can be heating the sample, cooling the sample, fluidizingthe sample, mixing the sample, stirring the sample, disrupting thesample, increasing permeability of a component of the sample, enhancinga reaction within the sample, and/or sterilizing the sample. Also, thedesired result can be an in vitro or an ex vivo treatment. This aspector any of the other aspects of the invention can include any or all ofthe following features. The method can further include the steps ofsensing at least one condition to which the sample is subjected duringprocessing and altering at least one of the sonic energy and thelocation of the sample in response to the sensed condition. During thesensing step, the sensed condition can be temperature, pressure, anoptical property, an altered chemical, an acoustic signal, and/or amechanical occurrence. During the altering step, the characteristic ofthe sonic energy that is altered can be waveform, duration ofapplication, intensity, and/or duty cycle. The method can furtherinclude the step of controlling temperature of the sample and canfurther include the step of controlling pressure to which the sample isexposed. During the step of exposing the sample to sonic energy, thesonic energy can be generated by spark discharges across a gap, laserpulses, piezoelectric pulses, electromagnetic shock waves,electrohydraulic shock waves, electrical discharges into a liquid,and/or chemical explosives. The sonic energy can be focused on thesample. The sample can contain a cell, and the method can furthercomprise the step of introducing a material into the cell. The materialcan be a polymer, an amino acid monomer, an amino acid chain, a protein,an enzyme, a nucleic acid monomer, a nucleic acid chain, a saccharide, apolysaccharide, an organic molecule, an inorganic molecule, a vector, aplasmid, and/or a virus. The method can further include the step ofextracting a component of the sample. During the controlling step, atleast one characteristic of the sonic energy is controlled, thatcharacteristic being waveform, duration of application, intensity, orduty cycle. The method can further include the step of the sampleflowing through a conduit. The sonic energy can include at least twodifferent frequencies, optionally in the form of a wavetrain. Thewavetrain can include a first wave component and a different second wavecomponent Alternatively or additionally, the wavetrain can include about1000 cycles per burst at about a 10% duty cycle at about a 500 mVamplitude.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The invention, in accordance with preferred and exemplaryembodiments, together with further advantages thereof, is moreparticularly described in the following detailed description, taken inconjunction with the accompanying drawings.

[0026] In the drawings, like reference characters generally refer to thesame parts throughout the different views. Also, the drawings are notnecessarily to scale, emphasis instead generally being placed uponillustrating principles of the invention.

[0027]FIG. 1 is a schematic illustration of one embodiment of theapparatus according to the invention;

[0028]FIG. 2 is a schematic illustration of one example of sonic energycontrol showing sine waves at a variable amplitude and frequency;

[0029]FIG. 3 is a schematic illustration of one example of anintra-sample positioning (dithering) profile showing height, heightstep, and radius;

[0030]FIG. 4A is a schematic illustration of a vertical-sided treatmentvessel;

[0031]FIG. 4B is a schematic illustration of a conical treatment vessel;

[0032]FIG. 4C is a schematic illustration of a curved treatment vessel;

[0033]FIGS. 5A-5C are schematic illustrations of several embodiments ofa treatment vessel with a combination of an upper and lower member andsamples in the vessels prior to treatment;

[0034]FIG. 6A is a schematic illustration of a treatment vesselpositioned over a collection container prior to transferring thecontents of the vessel to the container;.

[0035]FIG. 6B is a schematic illustration of a treatment vesselpositioned over a collection container after transferring some of thecontents of the vessel to the container;

[0036]FIG. 7 is a schematic illustration of an in-line fluid treatmentmethod in accordance with an alternative embodiment of the invention;

[0037]FIG. 8 is a graph depicting change in sample temperature as afunction of duty cycle at 500 mV and 750 mV, in one embodiment of theinvention;

[0038]FIG. 9 is a schematic illustration of an embodiment of theinvention with a microtiter plate containing samples, such that one ofthe wells of the microtiter plate is positioned at the focus point ofsonic energy;

[0039]FIG. 10 describes certain features and specifications related toperformance, consumables, procedure for treatment, and mechanicalcomponents of a system according to certain embodiments of theinvention;

[0040]FIG. 11 describes certain features and specifications related toinstrument control, user interface, electrical, and associated equipmentof a system according to certain embodiments of the invention;

[0041]FIG. 12 describes certain characteristics and functionality ofoperating software related to general functions, display functions,sonic energy control, and target/source positioning of a systemaccording to certain embodiments of the invention; and

[0042]FIG. 13 describes certain additional characteristics andfunctionality of operating software related to target/source positioningand temperature control of a system according to certain embodiments ofthe invention.

DETAILED DESCRIPTION OF THE INVENTION

[0043] “Sonic energy” as used herein is intended to encompass such termsas acoustic energy, acoustic waves, acoustic pulses, ultrasonic energy,ultrasonic waves, ultrasound, shock waves, sound energy, sound waves,sonic pulses, pulses, waves, or any other grammatical form of theseterms, as well as any other type of energy that has similarcharacteristics to sonic energy. “Focal zone” or “focal point” as usedherein means an area where sonic energy converges and/or impinges on atarget, although that area of convergence is not necessarily a singlefocused point. As used herein, the terms “microplate,” “microtiterplate,” “microwell plate,” and other grammatical forms of these termscan mean a plate that includes one or more wells into which samples maybe deposited. As used herein, “nonlinear acoustics” can mean lack ofproportionality between input and output. For example, in ourapplication, as the amplitude applied to the transducer increases, thesinusoidal output loses proportionality such that eventually the peakpositive pressure increases at a higher rate than the peak negativepressure. Also, water becomes nonlinear at high intensities, and in aconverging acoustic field, the waves become more disturbed as theintensity increases toward the focal point. Nonlinear acousticproperties of tissue can be useful in diagnostic and therapeuticapplications. As used herein, “acoustic streaming” can mean generationof fluid flow by acoustic waves. The effect can be non-linear. Bulkfluid flow of a liquid in the direction of the sound field can becreated as a result of momentum absorbed from the acoustic field. Asused herein, “acoustic microstreaming” can mean time-independentcirculation that occurs only in a small region of the fluid around asource or obstacle for example, an acoustically driven bubble in a soundfield. As used herein, “acoustic absorption” can refer to acharacteristic of a material relating to the material's ability toconvert acoustic energy into thermal energy. As used herein, “acousticimpedance” can mean a ratio of sound pressure on a surface to sound fluxthrough the surface, the ratio having a reactance and a resistancecomponent. As used herein, “acoustic lens” can mean a system or devicefor spreading or converging sounds waves. As used herein, “acousticscattering” can mean irregular and multi-directional reflection anddiffraction of sound waves produced by multiple reflecting surfaces, thedimensions of which are small compared to the wavelength, or by certaindiscontinuities in the medium through which the wave is propagated.

[0044] I. Apparatus and Methods for Ultrasonic Treatment

[0045] In certain embodiments, the apparatus includes a source of sonicenergy, a sensor for monitoring the energy or its effect, and a feedbackmechanism coupled with the source of sonic energy to regulate the energy(for example, voltage, frequency, pattern) for transmitting ultrasonicenergy to a target. Devices for transmission may include detection andfeedback circuits to control one or more of losses of energy atboundaries and in transit via reflection, dispersion, diffraction,absorption, dephasing and detuning. For example, these devices cancontrol energy according to known loss patterns, such as beam splitting.Sensors can detect the effects of ultrasonic energy on targets, forexample, by measuring electromagnetic emissions, typically in thevisible, IR, and UV ranges, optionally as a function of wavelength.These effects include energy dispersion, scattering, absorption, and/orfluorescence emission. Other measurable variables include electrostaticproperties such as conductivity, impedance, inductance, and/or themagnetic equivalents of these properties. Measurable parameters alsoinclude observation of physical uniformity, pattern analysis, andtemporal progression uniformity across an assembly of treatment vessels,such as a microtiter plate.

[0046] As shown in FIG. 1, one or more sensors coupled to a feedbackcontrol results in more focused, specific, or controlled treatment thanthat possible using current methods typical in the art. The feedbackmethodology can include fixed electronic elements, a processor, acomputer, and/or a program on a computer. The electronic elements,processor, computer, and/or computer program can in turn control any ofa variety of adjustable properties to selectively expose a sample tosonic energy in a given treatment. These properties can includemodulation of the ultrasonic beam in response to a detected effect.Modifiable ultrasonic wave variables can include intensity, duty cycle,pulse pattern, and spatial location. Typical input parameters that cantrigger an output can include change in level of signal, attainment ofcritical level, plateauing of effect, and/or rate of change. Typicaloutput actions can include sonic input to sample, such as frequency,intensity, duty cycle; stopping sample movement or sonic energy; and/ormoving beam within a sample or to the next sample.

[0047] More particularly, FIG. 1 depicts an electronically controlledultrasonic processing apparatus 100 that includes an ultrasoundtreatment system and associated electronics 200, a positioning system300 for the sample target 800 being treated, and a control system 400which controls, generates, and modulates the ultrasound signal andcontrols the positioning system 300 in a predetermined manner that mayor may not include a feedback mechanism. The source of sonic energy 230and the target 800 being treated for example, a sample, multiplesamples, or other device are arranged in a fluid bath 600, such aswater, such that the source of sonic energy 230 is oriented towards thetarget 800. The target 800 may be positioned proximate the surface ofthe fluid bath 600, above the source of sonic energy 230, all beingcontained within a sample processing vessel 500. Any of a multitude ofsensors 700 for measuring processing parameters can be arranged in orproximate to the fluid bath 600. A temperature control unit 610 may beused to control the temperature of the fluid in the fluid bath 610. Anoverpressure system 900 can control, for example, cavitation, bymaintaining a positive pressure on the target 800 and may be adjusted,in a predetermined manner that may or may not include feedbackprocessing, by a target pressure controller 910 that is connected to thecontrol system 400.

[0048] An ultrasound acoustic field 240 can be generated by the sonicenergy source 230, for example, a focused piezoelectric ultrasoundtransducer, into the fluid bath 600. According to one embodiment, thesonic energy source 230 can be a 70 mm diameter spherically focusedtransducer having a focal length of 63 mm, which generates anellipsoidal focal zone approximately 2 mm an diameter and 6 mm in axiallength when operated at a frequency of about 1 MHz. The sonic energysource 230 is positioned so that the focal zone is proximate the surfaceof the fluid bath 600. The sonic energy source 230 can be driven by analternating voltage electrical signal generated electronically by thecontrol system 400.

[0049] The positioning system 300 can include at least one motorizedlinear stage 330 that allows the target to be positioned according to aCartesian coordinate system. The positioning system 300 may position andmove the target 800 relative to the source 230 in three dimensions (x,y, z) and may optionally move either or both of the target 800 and thesonic energy source 230. The positioning system 300 can move target 800during and as part of the treatment process and between processes, aswhen multiple samples or devices within the target 800 are to beprocessed in an automated or high-throughput format. The positioningsystem 300 may position or move the target 800 in a plane transverse tothe focal axis of the sonic energy source 230 (x and y axes). Thepositioning system 300 can position and move the target 800 along thefocal axis of the sonic energy source 230 and lift or lower the target800 from or into the fluid bath 600 (z axis). The positioning system 300can also position the sonic energy source 230 and any or all of thesensors 700 in the fluid bath 600 along the focal axis of the sonicenergy source 230, if the sensors 700 are not affixed in the water bath600, as well as lift, lower, or otherwise move the sonic energy source230. The positioning system 300 also can be used to move other devicesand equipment such as detection devices and heat exchange devices fromor into the fluid bath 600 (z axis). The linear stages of thepositioning mechanism 330 can be actuated by stepper motors (not shown),which are driven and controlled by electrical signals generated by thecontrol system 400, or other apparatus known to those skilled in theart.

[0050] The control system 400 can include a computer 410 and a userinput/output device or devices 420 such as a keyboard, display, printer,etc. The control system is linked with the ultrasound treatment system200 to drive the sonic energy source 230, with the positioning system300 to drive the stepper motors described above, with one or moresensors 700 to detect and measure process conditions and parameters, andwith one or more controllers, such as the target pressure controller910, to alter conditions to which the target 800 is exposed. A fluidbath controller 610 could also be linked with the control system 400 toregulate temperature of the fluid bath 600. The user interface 420allows an operator to design and specify a process to be performed upona sample. In this regard, the ultrasound treatment system 200 caninclude an arbitrary waveform generator 210 that drives an RF amplifier220, such that the sonic energy source 230 receives an input. The outputsignal of the RF amplifier 220 may be conditioned by an impedancematching network and input to the sonic energy source 230. The computer410 also drives and controls the positioning system 300 through, forexample, a commercially available motion control board 310 and steppermotor power amplifier device 320.

[0051] The control system 400 can generate a variety of usefulalternating voltage waveforms to drive the sonic energy source 230. Forinstance, a high power “treatment” interval consisting of about 5 to1,000 sine waves, for example, at 1.1 MHz, may be followed by a lowpower “convection mixing” interval consisting of about 1,000 to1,000,000 sine waves, for example, at the same frequency. “Dead times”or quiescent intervals of about 100 microseconds to 100 milliseconds,for example, may be programmed to occur between the treatment andconvection mixing intervals. A combined waveform consisting ofconcatenated treatment intervals, convection mixing intervals, and deadtime intervals may be defined by the operator or selected from a storedset of preprogrammed waveforms. The selected waveform may be repeated aspecified number of times to achieve the desired treatment result.Measurable or discernible process attributes such as sample temperature,water bath temperature, intensity of acoustic cavitation, or visibleevidence of mixing in the sample processing vessel 500, may be monitoredby the control system 400 and employed in feedback loop to modifyautomatically the treatment waveform during the treatment process. Thismodification of the treatment waveform may be a proportional change toone or more of the waveform parameters or a substitution of onepreprogrammed waveform for another. For instance, if the sampletemperature deviates excessively during treatment from a set-pointtemperature due to absorbed acoustic energy, the control system 400 mayproportionally shorten the treatment interval and lengthen theconvection mixing interval in response to the error between the actualand target sample temperatures. Or, alternatively, the control system400 may substitute one predetermined waveform for another. The controlsystem 400 may be programmed to terminate a process when one or more ofthe sensors 700 signal that the desired process result has beenattained.

[0052] The control system 400 controls and drives the positioning system300 with the motion control board 310, power amplifier device 320, andmotorized stage 330, such that the target 800 can be positioned or movedduring treatment relative to the source 230 to selectively expose thetarget 800 to sonic energy, described more fully below.

[0053] Various aspects of the embodiment of FIG. 1 and of components ofthe embodiment shown in FIG. 1, as well as other embodiments with thesame, similar, and/or different components, are more fully describedbelow.

[0054] A. Transducer

[0055] In certain embodiments, the sonic energy source 230, for example,an ultrasound transducer or other transducer, produces acoustic waves inthe “ultrasonic” frequency range. Ultrasonic waves start at frequenciesabove those that are audible, typically about 20,000 Hz or 20 kHz, andcontinue into the region of megahertz (MHz) waves. The speed of sound inwater is about 1000 meters per second, and hence the wavelength of a1000 Hz wave in water is about a meter, typically too long for specificfocusing on individual areas less than one centimeter in diameter,although usable in non-focused field situations. At 20 kHz thewavelength is about 5 cm, which is effective in relatively smalltreatment vessels. Depending on the sample and vessel volume, preferredfrequencies may be higher, for example, about 100 kHz, about 1 MHz, orabout 10 MHz, with wavelengths, respectively, of approximately 1.0, 0.1,and 0.01 cm. In contrast, for conventional sonication, including sonicwelding, frequencies are typically approximately in the tens of kHz, andfor imaging, frequencies are more typically about 1 MHz and up to about20 MHz. In lithotripsy, repetition rates of pulses are fairly slow,being measured in the hertz range, but the sharpness of the pulsesgenerated give an effective pulse wavelength, or in this case, pulserise time, with frequency content up to about 100 to about 300 MHz, or0.1-0.3 gigahertz (GHz).

[0056] The frequency used in certain embodiments of the invention alsowill be influenced by the energy absorption characteristics of thesample or of the treatment vessel, for a particular frequency. To theextent that a particular frequency is better absorbed or preferentiallyabsorbed by the sample, it may be preferred. The energy can be deliveredin the form of short pulses or as a continuous field for a definedlength of time. The pulses can be bundled or regularly spaced.

[0057] A generally vertically oriented focused ultrasound beam may begenerated in several ways. For example, a single-element piezoelectrictransducer, such as those supplied by Sonic Concepts, Woodinville,Wash., that can be a 1.1 MHz focused single-element transducer, can havea spherical transmitting surface that is oriented such that the focalaxis is vertical. Another embodiment uses a flat unfocused transducerand an acoustic lens to focus the beam. Still another embodiment uses amulti-element transducer such as an annular array in conjunction withfocusing electronics to create the focused beam. The annular arraypotentially can reduce acoustic sidelobes near the focal point by meansof electronic apodizing, that is by reducing the acoustic energyintensity, either electronically or mechanically, at the periphery ofthe transducer. This result can be achieved mechanically by partiallyblocking the sound around the edges of a transducer or by reducing thepower to the outside elements of a multi-element transducer. Thisreduces sidelobes near the energy focus, and can be useful to reduceheating of the vessel. Alternatively, an array of small transducers canbe synchronized to create a converging beam. Still another embodimentcombines an unfocused transducer with a focusing acoustic mirror tocreate the focused beam. This embodiment can be advantageous at lowerfrequencies when the wavelengths are large relative to the size of thetransducer. The axis of the transducer of this embodiment can behorizontal and a shaped acoustic mirror used to reflect the acousticenergy vertically and focus the energy into a converging beam.

[0058] In certain embodiments, the focal zone can be small relative tothe dimensions of the treatment vessel to avoid heating of the treatmentvessel. In one embodiment, the focal zone has a radius of approximately1 mm and the treatment vessel has a radius of at least about 5 mm.Heating of the treatment vessel can be reduced by minimizing acousticsidelobes near the focal zone. Sidelobes are regions of high acousticintensity around the focal point formed by constructive interference ofconsecutive wavefronts. The sidelobes can be reduced by apodizing thetransducer either electronically, by operating the outer elements of amulti-element transducer at a lower power, or mechanically, by partiallyblocking the acoustic waves around the periphery of a single elementtransducer. Sidelobes may also be reduced by using short bursts, forexample in the range of about 3 to about 5 cycles in the treatmentprotocol.

[0059] The transducer can be formed of a piezoelectric material, such asa piezoelectric ceramic. The ceramic may be fabricated as a “dome”,which tends to focus the energy. One application of such materials is insound reproduction; however, as used herein, the frequency is generallymuch higher and the piezoelectric material would be typicallyoverdriven, that is driven by a voltage beyond the linear region ofmechanical response to voltage change, to sharpen the pulses. Typically,these domes have a longer focal length than that found in lithotripticsystems, for example, about 20 cm versus about 10 cm focal length.Ceramic domes can be damped to prevent ringing. The response is linearif not overdriven. The high-energy focus of one of these domes istypically cigar-shaped. At 1 MHz, the focal zone is about 6 cm long andabout 2 cm in diameter for a 20 cm dome, or about 15 mm long and about 3mm wide for a 10 cm dome. The peak positive pressure obtained from suchsystems is about 1 MPa (mega Pascal) to about 10 MPa pressure, or about150 PSI (pounds per square inch) to about 1500 PSI, depending on thedriving voltage.

[0060] The wavelength, or characteristic rise time multiplied by soundvelocity for a shock wave, is in the same general size range as a cell,for example about 10 to about 40 micron. This effective wavelength canbe varied by selection of the pulse time and amplitude, by the degree offocusing maintained through the interfaces between the source and thematerial to be treated, and the like.

[0061] In certain embodiments, the focused ultrasound beam is orientedvertically in a water tank so that the sample may be placed at or nearthe free surface. The ultrasound beam creates shock waves at the focalpoint. In an embodiment to treat industry standard microplates whichhold a plurality of samples in an array, a focal zone,.defined as havingan acoustic intensity within about 6 dB of the peak acoustic intensity,is formed around the geometric focal point. This focal zone has adiameter of approximately 2 mm and an axial length of about 6 mm.

[0062] Ceramic domes are adaptable for in vitro applications because oftheir small size. Also, systems utilizing ceramic domes can be producedat reasonable cost. They also facilitate scanning the sonic beam focusover a volume of liquid, by using microactuators which move a retainingplatform to which the sample treatment vessel is attached.

[0063] Another source of focused pressure waves is an electromagnetictransducer and a parabolic concentrator, as is used in lithotripsy. Theexcitation tends to be more energetic, with similar or larger focalregions. Strong focal peak negative pressures of about −16 MPa have beenobserved. Peak negative pressures of this magnitude provide a source ofcavitation bubbles in water, which can be desirable in an extractionprocess.

[0064] The examples described below use a commercial ultrasonic driverusing a piezoelectric ceramic, which is stimulated by application offluctuating voltages across its thickness to vibrate and so to produceacoustic waves. These may be of any of a range of frequencies, dependingon the size and composition of the driver. Such drivers are used inlithotripsy, for example, as well as in acoustic speakers and inultrasound diagnostic equipment, although without the control systems asdescribed herein.

[0065] These commercially-available drivers have a single focus.Therefore, to treat, for example, to stir, an entire microplate withsuch a device, it is typically necessary to sequentially position orstep each well at the focus of the driver. Because stirring time isbrief, the stepping of a 96 well plate can be accomplished inapproximately two minutes or less with simple automatic controls, asdescribed below. It is contemplated that this time can be shortened.

[0066] It also is possible to make multi-focal drivers by makingpiezoelectric devices with more complex shapes. Modulators of theacoustic field attached to an existing piezoelectric driver can alsoproduce multiple foci. These devices can be important for obtainingrapid throughput of microplates in a high density format, such as the1534-well format.

[0067] B. Drive Electronics and Waveform Control.

[0068] One treatment protocol can include variable acoustic waveformscombined with sample motion and positioning to achieve a desired effect.The acoustic waveform of the transducer has many effects, including:acoustic microstreaming in and near cells due to cavitation, that isflow induced by, for example, collapse of cavitation bubbles; shockwaves due to nonlinear characteristics of the fluid bath; shock wavesdue to cavitation bubbles; thermal effects, which lead to heating of thesample, heating of the sample vessel, and/or convective heat transferdue to acoustic streaming; flow effects, causing deflection of samplematerial from the focal zone due to shear and acoustic pressure, as wellas mixing due to acoustic streaming, that is flow induced by acousticpressure; and chemical effects.

[0069] The treatment protocol can be optimized to maximize energytransfer while minimizing thermal effects. The treatment protocol alsocan effectively mix the contents of the treatment vessel, in the case ofa particulate sample suspended in a liquid. Energy transfer into thesample can be controlled by adjusting the parameters of the acousticwave such as frequency, amplitude, and cycles per burst. Temperaturerise in the sample can be controlled by limiting the duty cycle of thetreatment and by optimizing heat transfer between the treatment vesseland the water bath. Heat transfer can be enhanced by making thetreatment vessel with thin walls, of a relatively highly thermallyconductive material, and/or by promoting forced convection by acousticstreaming in the treatment vessel and in the fluid bath in the proximityof the treatment vessel. Monitoring and control of temperature isdiscussed in more detail below.

[0070] For example, for a cellular disruption and extraction treatment,an example of an effective energy waveform is a high amplitude sine waveof about 1000 cycles followed by a dead time of about 9000 cycles, whichis about a 10% duty cycle, at a frequency of about 1.1 MHz. The sinewave electrical input to the transducer typically results in a sine waveacoustic output from the transducer. As the focused sine waves convergeat the focal point, they can become a series of shock waves due to thenonlinear acoustic properties of the water or other fluid in the bath.This protocol treats the material in the focal zone effectively duringthe “on” time. As the material is treated, it typically is expelled fromthe focal zone by acoustic shear and streaming. New material circulatesinto the focal zone during the “off” time. This protocol can beeffective, for example, for extracting the cellular contents of groundor particulate leaf tissue, while causing minimal temperature rise inthe treatment vessel.

[0071] Further advantage in disruption and other processes may be gainedby creating a high power “treat” interval 10 alternating with a lowpower “mix” interval 14, as shown schematically in FIG. 2. Moreparticularly, in this example, the “treat” interval 10 utilizes a sinewave that has a treatment frequency 18, a treatment cycles-per-burstcount 26, and a treatment peak-to-peak amplitude 22. The “mix” interval14 has a mix frequency 20, a mix cycles-per-burst count 28 and a lowermix peak-to-peak amplitude 24. Following each of the intervals 10, 14 isa dead time 12, 16. Of course, these relationships are merely oneexample of many, where one interval in considered to be high power andone interval is considered to be low power, and these variables andothers can be altered to produce more or less energetic situations.Additionally, the treat function or interval and the mix function orinterval could emit from different or multiple transducers in the sameapparatus, optionally emitting at different frequencies.

[0072] High power/low power interval treatments can allow multipleoperations to be performed, such as altering permeability of components,such as cells, within the sample followed by subsequent mixing of thesample. The treat interval can maximize cavitation and bioeffects, whilethe mix interval can maximize mixing within the treatment vessel and/orgenerate minimal heat. Adding a longer, high power “super-mix” intervaloccasionally to stir up particles that are trapped around the peripheryof the treatment vessel can provide further benefits. This “super-mix”interval generates additional heat, so it is programmed to treatinfrequently during the process, for example, every few seconds.Additionally, dead times between the mix and treat intervals, duringwhich time substantially no energy is emitted from the sonic energysource, can allow fresh material to circulate into the energy focal zoneof the target.

[0073] As discussed below, moving the sample vessel during treatmentrelative to the source, so that the focal zone moves within thetreatment vessel, can further enhance the process. For example, targetmotion through the focal zone can resuspend material in the sample thatmay have clumped or become trapped around the periphery of the treatmentvessel. A similar improvement can be achieved by traversing or“dithering” the treatment vessel relative to the focal zone, describedmore fully below with respect to FIG. 3. Dithering can becomeincreasingly advantageous as the sample treatment vessel becomessignificantly larger than the focal zone.

[0074] The waveform of focused sound waves can be a single shock wavepulse, a series of individual shock wave pulses, a series of shock wavebursts of several cycles each, or a continuous waveform. Incidentwaveforms can be focused directly by either a single element, such as afocused ceramic piezoelectric ultrasonic transducer, or by an array ofelements with their paths converging to a focus. Alternatively, multiplefoci can be produced to provide ultrasonic treatment to multipletreatment zones, vessels, or wells.

[0075] Reflected waveforms can be focused with a parabolic reflector,such as s used in an “electromagnetic” or spark-gap type shock-wavegenerator. Incident and reflected waveforms can be directed and focusedwith an ellipsoidal reflector such as is used in an electrohydraulicgenerator. Waveforms also can be channeled.

[0076] The waveform of the sound wave typically is selected for theparticular material being treated. For example, to enhance cavitation,it can be desirable to increase the peak negative pressure following thepeak positive pressure. For other applications, it can be desirable toreduce cavitation but maintain the peak positive pressure. This resultcan be achieved by performing the process in a pressurized chamber at aslight pressure above ambient. For example, if the waveform generatedhas a peak negative pressure of about −5 MPa, then the entire chambermay be pressurized to about 10 MPa to eliminate cavitation fromoccurring during the process. Liquid to be treated can be pressurized ona batch or a continuous basis.

[0077] A variety of methods of generating waves can be used. Inlithotripsy, for example, “sharp” shock waves of high intensity andshort duration are generated. Shock waves may be generated by any methodthat is applicable to a small scale. Such methods include sparkdischarges across a known gap; laser pulses impinging on an absorptiveor reflective surface; piezoelectric pulses; electromagnetic shockwaves; electrohydraulic shock waves created by electrical discharges ina liquid medium; and chemical explosives. In the case of explosives,microexplosives in wells in a semiconductor-type chip can be fabricatedin which the wells are individually addressable. Also, amagnetostrictive material can be exposed to a magnetic field, and it canexpand and/or contract such that the material expansion/contractioncreates sonic energy.

[0078] Continuous sinusoidal sound waves can be generated by any processthat is appropriate for focusing on a small scale. For example, ceramicpiezoelectric elements may be constructed into dome shapes to focus thesound wave into a point source. In addition, two or more shock waves maybe combined from the same source, such as piezoelectric elementsarranged in mosaic form, or from different sources, such as anelectromagnetic source used in combination with a piezoelectric source,to provide a focused shock wave.

[0079] Typically, the shock wave is characterized by a rapid shock frontwith a positive peak pressure in the range of about 15 MPa, and anegative peak pressure in the range of about negative 5 MPa. Thiswaveform is of about a few microseconds duration, such as about 5microseconds. If the negative peak is greater than about 1 MPa,cavitation bubbles may form. Cavitation bubble formation also isdependent upon the surrounding medium. For example, glycerol is acavitation inhibitive medium, whereas liquid water is a cavitationpromotive medium. The collapse of cavitation bubbles forms “microjets”and turbulence that impinge on the surrounding material.

[0080] The waves are applied to the samples either directly, as forexample, piezoelectric pulses, or via an intervening medium. This mediumcan be water or other fluid. An intervening medium also can be a solid,such as a material which is intrinsically solid or a frozen solution.Waves also can be applied through a container, such as a bottle, bag,box, jar, or vial.

[0081] For maximum control, and particularly for well-by-well mixing, afocused acoustic pulse is useful. When a pulse is emitted from a curvedsource with an elliptical profile, then the emitted acoustic waves orpulses focus in a small region of maximum intensity. The location of thefocus can be calculated or determined readily by experiment. Thediameter of the focal zone can be of the same general size as or smallerthan the diameter of the treatment vessel. Then, mixing energy can beprovided to each well for a repeatable amount of time, providing uniformmixing of each sample.

[0082] C. X-Y-Z Cartesian Positioning System.

[0083] In certain embodiments, the sample is not only moved intoposition relative to the transducer initially, but positioned duringtreatment to insure uniform treatment of the sample, where the sample iskept well suspended during treatment. As used herein, x and y axesdefine a plane that is substantially horizontal relative to groundand/or a base of an apparatus of the invention, while the z axis lies ina plane that is substantially vertical relative to the ground and/or thebase of an apparatus and perpendicular to the x-y plane.

[0084] One positioning scheme is termed “dithering,” which entailsslightly varying the position of the sample relative to the source whichcan occur by moving the sample through the focal zone in several ways.For example, but without limitation, the sample can be moved in acircle, or oval, or other arcuate path with a certain radius 30 andmoved a certain distance 34 in certain increments or steps 32, asdepicted schematically in FIG. 3. These movements can vary betweentreatment cycles or during a particular treatment cycle and have severaleffects. First, dithering the sample position sweeps the focal zonethrough the volume of the sample treatment vessel or device, treatingmaterial that is not initially in the focal zone. In addition, varyingthe location of the acoustic focus within the vessel tends to maketreatment, and the resulting heating, more uniform within each sample.

[0085] Certain embodiments include drive electronics and devices forpositioning of the sample(s). In one embodiment, the positioningsequence, optionally including dithering, and the treatment pulse trainare pre-programmed, for example in a computer, and are executedautomatically. The driver electronics and positioners can be linkedthrough the control system to sensors so that there is “real time”feedback of sensor data to the control system during treatment in orderto adjust the device(s) for positioning the sample and prevent localizedheating or cavitation. The drive electronics can include a waveformgenerator matching network, an RF switch or relay, and a radio frequency(RF) amplifier, for safety shutdown.

[0086] The positioning system can include a three axis Cartesianpositioning and motion control system to position the sample treatmentvessel or an array of sample treatment vessels relative to theultrasound transducer. The “x” and “y” axes of the Cartesian positioningsystem allow each sample in an array of samples, such as an industrystandard microplate, to be brought into the focal zone for treatment.Alternative configurations may employ a combination of linear and rotarymotion control elements to achieve the same capabilities as the threeaxis Cartesian system. Alternative positioning systems may beconstructed of self-contained motor-driven linear or rotary motionelements mounted to each other and to a base plate to achieve two- orthree-dimensional motion.

[0087] As used in the examples, stepper motors, such as those availablefrom Eastern Air Devices, located in Dover, N.H., drive linear motionelements through lead screws to position the sample. The stepper motorsare driven and controlled by means of LabVIEW software controlling aValueMotion stepper motor control board available from NationalInstruments, located in Austin, Tex. The output signals from the controlboard are amplified by a nuDrive multi-axis power amplifier interface,also available from National Instruments, to drive the stepper motors.

[0088] The computer controlled positioning system can be programmed tosequentially move any defined array of multiple samples into alignmentwith the focal zone of the ultrasound transducer. If temperature riseduring treatment is an issue, the samples in a multi-sample array can bepartially treated and allowed to cool as the positioning systemprocesses the other samples. This can be repeated until all the sampleshave been treated fully.

[0089] The positioning system also can move the sample treatment vesselrelative to the focal point during treatment to enhance the treatment orto treat a sample that is large relative to the focal zone. By sweepingthe sample slowly in a circular or other motion during treatment, clumpsof material around the periphery of the treatment vessel may be brokenup advantageously. In-addition, x-y dithering may prevent a “bubbleshield” from forming and blocking cavitation in the sample treatmentvessel. The x-y dithering may also enhance treatment of samplesuspensions that have a high viscosity or become more viscous duringtreatment and do not mix well. The sample position may also be ditheredvertically in the Z axis. This may be advantageous in a deep treatmentvessel where the depth is substantially larger than the axial dimensionof the focal zone, in order to treat the entire contents of thetreatment vessel or to resuspend larger sample fragments which have sunkto the bottom of the vessel. Dithering in all three dimensions may alsobe employed, as depicted in FIG. 3

[0090] For a relatively flat sample, such as whole leaf tissue, ahistological sample, or thin-section specimen, where the area of thesample is large relative to the cross-sectional area of the focal zone,the x-y positioning system can cause the focal zone to traverse thesample in order to treat the entire surface of the sample. Thisprocedure may be combined with optical analysis or other sensors todetermine the extent of the treatment to each portion of the sample thatis brought into the focal zone.

[0091] In certain embodiments, the sample or array of samples can bemoved relative to the transducer and the other parts of the apparatus.In alternative embodiments the transducer is moved while the sampleholder remains fixed, relative to the other parts of the apparatus. Asan alternative, movement along two of the axes, for example, x and y,can be assigned to the sample holder and movement along the third axis,z in this case, can be assigned to the transducer.

[0092] The three axis positioning system enables automated energy focusadjustment in the z axis when used in conjunction with a sensor formeasuring the ultrasound intensity. In one embodiment, a needlehydrophone can be mounted in a fixture on the sample positioning system.The hydrophone can be traversed in three dimensions through the focalregion to record the acoustic intensity as a function of position inorder to map out the focal zone. In another embodiment, a number ofpositions on a sheet of aluminum foil held in the sample holder can betreated in a sequence of z-axis settings. The foil can then be examinedto determine the spot size of the damage at each position. The diameterof the spot corresponds generally to the diameter of the focal zone atthat z-axis setting. Other, fully automated embodiments of a focusingsystem can also be constructed.

[0093] The three axis positioning system also allows the apparatus to beintegrated into a larger laboratory automation scheme. A positioningsystem with an extended work envelope can transfer microplates or othersample vessels into and out of the apparatus. This allows the apparatusto interact automatically with upstream and downstream processes.

[0094] D. Sensors

[0095] Visual Monitoring of the Sample

[0096] Optical or video detection and analysis can be employed tooptimize treatment of the sample. For example, in a suspension ofbiological tissue, the viscosity of the mixture can increase duringtreatment due to the diminution of the particles by the treatment and/orby the liberation of macromolecules into the solution. Video analysis ofthe sample during treatment allows an automated assessment of the mixingcaused by the treatment protocol. The protocol may be modified duringthe treatment to promote greater mixing as a result of this assessment.The video data may be acquired and analyzed by the computer controlsystem that is controlling the treatment process. Other opticalmeasurements such as spectral excitation, absorption, fluorescence,emission, and spectral analysis also can be used to monitor treatment ofthe sample. A laser beam, for example, can be used for alignment and toindicate current sample position.

[0097] Monitoring of Temperature

[0098] Heating of individual wells can be determined by an infraredtemperature-sensing probe, collimated so as to view only the well beingtreated with the ultrasonic energy. For example, an infrared thermalmeasuring device can be directed at the top unwetted side of thetreatment vessel. This provides a non-contact means of analysis that isnot readily achievable in conventional ultrasound treatmentconfigurations. The thermal information can be recorded as a thermalrecord of the sample temperature profile during treatment.

[0099] Active temperature monitoring may be used as a feedback mechanismto modify the treatment protocol during the treatment process to keepthe sample temperature within specified limits. For example, an infraredsensor directed at the sample treatment vessel may input temperaturereadings to the computer. The computer, in accordance with a controllingprogram, can produce output directed to the circuit enabling-theultrasonic transducer, which in turn can reduce the high power treatmentintervals and increase the low power mixing intervals, for example, ifthe sample temperature is nearing a specified maximum temperature.

[0100] Monitoring of Cavitation

[0101] A variety of methods may be employed to detect cavitation. Forexample, acoustic emissions, optical scattering, high-speed photography,mechanical damage, and sonochemicals can be used. As described above formonitoring temperature, information from cavitation detection can beused by the system to produce an output that selectively controlsexposure of a sample to sonic energy in response to the information.Each of these methods to monitor cavitation are described more fullybelow.

[0102] Acoustic emissions: Bubbles are effective scatterers ofultrasound. The pulsation mode of a bubble is referred to as monopolesource, which is an effective acoustic source. For small, generallylinear oscillations, the bubble simply scatters the incident acousticpulse. However, as the response becomes more nonlinear, it also startsto emit signals at higher harmonics. When driven harder, the bubblesstart to generate subharmonics as well. Eventually as the responsebecomes a periodic or chaotic, the scattered field tends towards whitenoise. In the scenario where inertial collapses occur, short acousticpressure pulses are emitted. An acoustic transducer can be configured todetect these emissions. There is a detectable correlation between theonset of the emissions and cell disruption.

[0103] Optical scattering: Bubbles also scatter light. When bubbles arepresent, light is scattered. Light can normally be introduced into thesystem using fiber optic light sources so that cavitation can bedetected in real-time, and therefore can be controlled by electronic andcomputer systems.

[0104] High-speed photography: Bubbles can be photographed. This methodtypically requires high-speed cameras and high intensity lighting,because the bubbles respond on the time frame of the acoustics. It alsorequires good optical access to the sample under study. This method cangive detailed and accurate data and may be a consideration whendesigning systems according to the invention. Stroboscopic systems,which take images far less frequently, can often give similarqualitative performance more cheaply and easily than high-speedphotography.

[0105] Mechanical damage: Cavitation is known to create damage tomechanical systems. Pitting of metal foils is a particularly commoneffect, and detection method. There is a correlation between thecavitation needed to pit foils and to disrupt cells.

[0106] Sonochemicals: A number of chemicals are known to be produced inresponse to cavitation. The yield of these chemicals can be used as ameasure of cavitational activity. A common technique is to monitor lightgeneration from chemicals, such as luminol, that generate light whenexposed to cavitation. Sonochemical yield usually can not be done duringcell experiments but can be done independently under identicalconditions, and thereby, provide a calibrated standard.

[0107] E. Temperature, Cavitation, and Pressure Management and Control.

[0108] Temperature Control

[0109] Certain applications require that the temperature of the samplebeing processed be managed and controlled during processing. Forexample, many biological samples should not be heated above 4° C. duringtreatment. Other applications require that the samples be maintained ata certain elevated temperature during treatment. The ultrasoundtreatment protocol influences the sample temperature in several ways:the sample absorbs acoustic energy and converts it to heat; the sampletreatment vessel absorbs acoustic energy and converts it to heat which,in turn, can heat the sample; and acoustic streaming develops within thesample treatment vessel and the water bath, forcing convective heattransfer between the sample treatment vessel and the water bath. In thecase of a relatively cool water bath, this cools the sample.

[0110] The acoustic waves or pulses can be used to regulate thetemperature of the solutions in the treatment vessel. At low power, theacoustic energy produces a slow stirring without marked heating.Although energy is absorbed to induce the stirring, heat is lost rapidlythrough the sides of the treatment vessel, resulting in a negligibleequilibrium temperature increase in the sample. At higher energies, moreenergy is absorbed, and the temperature rises. The degree of rise perunit energy input can be influenced and/or controlled by severalcharacteristics, including the degree of heat absorption by the sampleor the treatment vessel and the rate of heat transfer from the treatmentvessel to the surroundings. Additionally, the treatment protocol mayalternate a high-powered treatment interval, in which the desiredeffects are obtained, with a low power mixing interval, in whichacoustic streaming and convection are achieved without significant heatgeneration. This convection may be used to promote efficient heatexchange or cooling.

[0111] The thermal information can also be used to modify or control thetreatment to maintain the sample temperature rise below a maximumallowable value. The treatment can be interrupted to allow the sample tocool down. In certain embodiments, the output of the thermal measurementdevice or system is entered into the computer control system forrecording, display on a control console, and/or control of exposure ofthe sample to sonic energy through a feedback loop, for example byaltering the duty cycle.

[0112] Temperature rise during ultrasonic continuous wave exposure canbe controlled, if required, by refrigeration of a liquid or other samplebefore, during, or after passage through a zone of sonic energy, ifprocessing in a continuous, flow-through mode. In generally stationarydiscrete sample processing modes, a sample can be cooled by air, bycontact with a liquid bath, or a combination of both air and liquid. Thetemperature is rapidly equilibrated within the vessel by the stirringaction induced by the acoustic waves. As a result, and especially insmall vessels or other small fluid samples, the rate of temperatureincrease and subsequent cooling can be very rapid. The rate of deliveryof sonic energy to the material can also be controlled, although thatcan lengthen processing time.

[0113] Liquids within the sample can be provided at any temperaturecompatible with the process. The liquid may be frozen or partiallyfrozen for processing. For example, when biological material issubjected to subzero temperatures below about −5° C., most, but not all,of the water is in the solid phase. However, in certain biologicaltissues, micro-domains of liquid water still remain for several reasons,such as natural “antifreeze” molecules or regions of higher saltconcentration. Therefore, sample temperature may be varied during theprocedure. A temperature is selected at which microdomains of liquidwater are able to form shock wave induced cavitation due to bubbleformation and collapse, resulting in shear stresses that impinge onsurrounding tissues. Indeed, gradually altering the sample temperaturecan be desirable, as it provides focused domains of liquid water forcollection of sonic energy for impingement on the surrounding material.

[0114] Treatment baths can be relatively simple, and can include a waterbath or other fluid bath that is employed to conduct the acoustic wavesfrom the transducer to the sample treatment vessel, where the liquid istemperature controlled. In certain embodiments, the entire bath ismaintained at a specific temperature by means of an external heater orchiller, such as a Neslab RTE-210 chiller available from NeslabInstruments, Inc., located in Newington, N.H., and heat exchanger coilsimmersed in the bath. The sides and bottom of the tank containing thebath may have sufficient insulating properties to allow the bath to bemaintained substantially uniformly at a specific temperature. Anotherembodiment, such as that depicted in FIG. 9, employs an inner tray orsample tank 76 made of an insulating material such as rigid polystyrenefoam which is set within a larger water bath 84 in a transducer tank 82.The inner tray 76 has heat-exchanger tubes or other heating or coolingdevices within it (not shown) to allow a fluid 78 such as ethyleneglycol or propylene glycol in the inner tray 76 to be heated or cooledbeyond what may be practical for the fluid 84 such as water in the outerbath in the transducer tank 82. The inner tray 76 has an acoustic window88 in the bottom. The acoustic window 88 is made of a thin film materialhaving low acoustic absorption and an acoustic impedance similar towater. This inner tray 76 is arranged so that the acoustic window 88 isaligned with a transducer 86 which is outside the tray 76, supportedwith a support 80 in the water 84. A sample 74 is located within amicrotiter plate or other sample treatment vessel 72, within the tray 76and is subjected to the thermal influence of the inner treatment bath78. The treatment vessel 70 can be movable relative to the transducer 86with a positioning system 70. Also, sonic energy focuses on the sample74 through the acoustic window 88. This arrangement permits the use ofseparate fluids and substantially independent control of the temperatureof the inner 76 and outer treatment baths 84. The smaller volume of theinner tray 76 facilitates the use of antifreeze mixtures, such as amixture of propylene glycol and water, at temperatures below thefreezing temperature of water. This, in turn, allows the samples 74 tobe processed and treated at temperatures below the freezing temperatureof water. This embodiment is beneficial for treatment applicationsrequiring that the sample materials 74 be maintained at temperaturesnear or below the freezing point of water. It allows for the containmentof treatment bath fluids 78, such as antifreeze solutions, that may notbe compatible with the transducer 86 and other system components. Italso allows the transducer 86 to be maintained at a differenttemperature than the samples 74. This embodiment may also be connectedwith any of the other components described in FIG. 1 and is suitable foruse in a system with or without feedback loop control.

[0115] Sample temperature may be required to remain within a giventemperature range during a treatment procedure. Temperature can bemonitored remotely by, for example, an infra-red sensor. Temperatureprobes such as thermocouples may not be particularly well suited for allapplications because the sound beam may interact with the thermocoupleand generate an artificially high temperature in the vicinity of theprobe. Temperature can be monitored by the same computer that controlsacoustic waveform. The control responds to an error signal which is thedifference between the measured actual temperature of the sample and thetarget temperature of the sample. The control algorithm can be as ahysteritic bang-bang controller, such as those in kitchen stoves, where,as an output of the control system, the acoustic energy is turned offwhen the actual temperature exceeds a first target temperature andturned on when the actual temperature falls below a second targettemperature that is lower than the first target temperature. Morecomplicated controllers can be implemented. For example, rather thansimply turning the acoustic signal on and off, the acoustic signal couldcontinuously be modulated proportionally to the error signal, forexample, by varying the amplitude or the duty cycle, to provide finertemperature regulation.

[0116] In the application of a bang-bang control algorithm for amultiple sample format, once a maximum temperature value has beenexceeded and the sonic energy is turned off for a particular sample, analternative to waiting for the sample to cool below a selectedtemperature before turning the sonic energy on again, is to move on tothe next sample. More particularly, some of the samples can be at leastpartially treated with sonic energy, in a sequence, and then, the systemcan return to the previously partially treated samples to take a sensorreading to determine if the samples have cooled below the selectedtemperature and to reinitiate treatment if they have. This proceduretreats the samples in an efficient manner and reduces the total itreatment time for treating multiple samples. Another alternative is toswitch to a predefined “cooling” waveform which promotes convectionwithout adding significant heat to a particular sample, rather thanmoving on to the next sample and returning to the first sample at alater time.

[0117] If uniformity of temperature throughout the sample is important,then control techniques can be used to ensure a uniform temperaturedistribution. An array of infra-red sensors can be used to determine thedistribution of the temperature inside the sample. If areas of increasedtemperature relative to the rest of the sample appear, then thetransducer can be switched from high power “treatment” mode to low power“mixing” mode. In the low power “mixing” mode, the sample isacoustically stirred until the sample is substantially uniform intemperature. Once temperature uniformity is achieved, the high power“treatment” mode is reinitiated. A control system can monitortemperature and responsively turn the various modes on or off. Whencontrolled by a computer, the intervals during which these modes areused can be very short, for example fractions of a second, thereby notsignificantly prolonging treatment times. Stepping times between wells,or other sample containers, can also be less than a second with suitabledesign.

[0118] Cavitation Control

[0119] In some applications, it can be preferable to treat the samplewith as much energy as possible without causing cavitation. This resultcan be achieved by suppressing cavitation. Cavitation can be suppressedby pressurizing the treatment vessel above ambient, often known as“overpressure,” to the point at which no negative pressure developsduring the rarefaction phase of the acoustic wave. This suppression ofcavitation is beneficial in applications such as cell transformationwhere the desired effect is to open cellular membranes while maintainingviable cells. In other applications it may be desirable to enhancecavitation. In these applications, a “negative” overpressure or vacuumcan be applied to the region of the focal zone.

[0120] The control of cavitation in the sample also can be importantduring acoustic treatment processes. In some scenarios, the presence ofsmall amounts of cavitation may be desirable to enhance biochemicalprocesses; however, when large numbers of cavitation bubbles exist theycan scatter sound before it reaches the target, effectively shieldingthe sample.

[0121] Cavitation can be detected by a variety of methods, includingacoustic and optical methods. An example of acoustic detection is apassive cavitation detector (PCD) which includes an external transducerthat detects acoustic emissions from cavitation bubbles. The signal fromthe PCD can be filtered, for example using a peak detector followed by alow pass filter, and then input to a controlling computer as a measureof cavitation activity. The acoustic signal could be adjusted in wayssimilar to those described in the temperature control example tomaintain cavitation activity at a desired level.

[0122] Overpressure: Increased ambient pressure is one technique forcontrolling cavitation. Overpressure tends to remove cavitation nuclei.Motes in the fluid are strongly affected by overpressure and socavitation in free-fluid is often dramatically reduced, even by theaddition of one atmosphere of overpressure. Nucleation sites oncontainer walls tend to be more resistant to overpressure; however thecavitation tends to be restricted to these sites and any gas bubblesthat float free into the free-fluid are quickly dissolved. Thereforecells in the bulk fluid are typically unaffected by cavitation sitesrestricted to the container walls. Overpressure may be applied to thetreatment vessel, the array of treatment vessels, the treatment bath andtank, or to the entire apparatus to achieve a higher than atmosphericpressure in the region of the focal zone.

[0123] Degassing: Reducing the gas content of the fluid tends to reducecavitation, again by reducing cavitation nuclei and making it harder toinitiate cavitation. Another method of controlling cavitation or theeffects of cavitation is to control the gasses that are dissolved in thesample fluid. For instance, cavitation causes less mechanical damage influid saturated with helium gas than in fluid saturated with argon gas.

[0124] Filtering: Cleaner fluids tend to be harder to cavitate.

[0125] Various fluids: Certain fluids are much harder to cavitate.Castor oil and mineral oil are nearly cavitation free. Two possiblereasons are that the fluids are of a nature that they tend to fill incracks, and that their viscosity also makes them more resistant tocavitation. The fluids, however, are not particularly compatible withcell preparations.

[0126] Waveform shape: The cavitation field responds to the acousticdriving pulse. It is possible to control the cavitation response, tosome extent, by controlling the driving acoustic pressure. Cavitationmay also be reduced or eliminated by reducing the number of cycles ineach burst of acoustic energy. The cavitation bubbles grow over severalcycles then collapse creating cavitation effects. By limiting the numberof cycles in each burst, bubble growth and collapse can be substantiallyavoided.

[0127] F. Treatment or Reaction Vessel

[0128] Treatment vessels are sized and shaped as appropriate for thematerial to be treated. They can be any of a variety of shapes. Forexample, as shown in FIGS. 4A-4C, treatment vessels 502, 504, 506 canhave vertical walls, can have a conical shape, or can have a curvedshape, respectively. As shown in FIGS. 5A-5C, certain treatment vessel502, 506, prior to treatment with sonic energy, have an upper member 530and a lower member 550 which together form an interior region thatcontains the material 540 to be treated. In certain embodiments, theultrasound transducer projects a focused ultrasound beam upwards. Theultrasound beam penetrates the lower member 550 of the treatment vessel.502, 506 to act upon the contents 540 of the treatment vessel 502, 506.The upper member 530 serves to contain the contents 540 of the vessel502, 506.

[0129] The lower member 550 of the treatment vessel 502, 506 isconfigured to transmit the maximum amount of ultrasound energy to thecontents 540 of the vessel 502, 506, minimize the absorption ofultrasound energy within the walls of the vessel 502, 506 and maximizeheat transfer between the contents 540 of the treatment vessel 502, 506and, for example, an external water bath. In certain embodiment of thepre-treatment assembly, the treatment vessel is thermoformed from a thinfilm in a hemispherical shape. The film should have an acousticimpedance similar to that of water and low acoustic absorption. Onepreferred material is low density polyethylene. Alternative materialsinclude polypropylene, polystyrene, poly(ethylene teraphthalate)(“PET”), and other rigid and flexible polymers. The film may be alaminate to facilitate thermal bonding, for example using heat sealing.Thicker, more rigid materials may also be employed. Available multi-wellplates in industry standard formats such as 96 well and 24 well formatsmay be employed with or without modification. Industry standardthick-wall, multi-well plates with thin film bottoms may also beemployed. These can work particularly advantageously where the size ofthe focal zone of the ultrasound beam is smaller than a well. In thiscase, little energy is absorbed by the sides of the treatment vesseland, as a result, relatively little energy is converted to heat.

[0130] The upper member of the treatment vessel contains the contents inthe vessel during treatment and can act also as an environmental seal.The upper member of the treatment vessel can be flat or domed to enclosethe interior of the treatment vessel. The upper member of the treatmentvessel may be made of a rigid or flexible material. Preferably, thematerial will have low acoustic absorption and good heat transferproperties. In certain embodiments of the pre-treatment assembly, theupper member of the treatment vessel is a thin film that can be bondedto the lower member, and the lower or upper member can be easilyrupturable for post-treatment transfer of the treated material.

[0131] The upper and lower members of the treatment vessel may be joinedtogether by thermal bonding, adhesive bonding, or external clamping.Such joining of the upper and lower members can serve to seal thecontents of the vessel from contaminants in the external environmentand, in an array of vessels, prevent cross-contamination betweenvessels. If the bond is to be achieved by thermal bonding, the upper andlower members of the treatment vessels may be made of film laminateshaving heat bondable outer layers and heat resistant inner layers.

[0132] The treatment vessel may be configured as a single unit, as amultiplicity of vessels in an array, or as a single unit with variouscompartments. The upper and lower members of the vessel or array ofvessels can be used once or repeatedly. There also can be a separateframe or structure (not shown) that supports and/or stiffens the upperand lower members of the vessel(s). This frame or structure may beintegral with the vessels or may be a separate member. An array oftreatment vessels may be configured to match industry standardmulti-well plates. In one embodiment the treatment vessel is configuredin an array that matches standard 96 well or 24 well multi-well plates.The frame or supporting structure holding the array of treatment vesselscan have the same configuration and dimensions as standard multi-wellplates.

[0133] As shown in FIGS. 6A and 6B, a treatment vessel 508 can include afunnel 592 to facilitate transfer of the contents 540 from the treatmentvessel 508 to a separate vessel 598 after treatment. The funnel 592 canhave a conical shape and include an opening at the narrow end. Thefunnel 592 can be rigid, relative to the upper 530 and lower members 550of the treatment vessel 508. The large end of the funnel 592 isproximate the upper member 550 of the treatment vessel 508 and alignedwith the treatment vessel 508. The volume of the funnel 592 can bemarginally less than the volume of the treatment vessel 508.

[0134] One process of transferring the contents 540 of the treatmentvessel 508 to another post-treatment vessel 598 includes the followingsteps. The upper member 530 of the treatment vessel 508 may be piercedwith a sharp instrument or ruptured when a vacuum is applied. Tofacilitate rupture, the member 530 may be manufactured from a thinfragile material or made weak by etching a feature into the surface.Then, the treatment vessel 508 is inverted over the post-treatmentvessel 598 in a vacuum fixture. A filter 594 may be placed between thetreatment vessel 508 and the post-treatment vessel 598 to separatesolids 596 from the liquid 542 that is removed from the treatment vessel508. Alternatively, the filter 594 may be incorporated into the outletof the funnel 592. This arrangement of treatment vessel 508 and funnel592 may be configured as a single unit or as an array of units. Thisarray may match an industry standard. The treatment vessel 508 shouldform a vacuum seal with a vacuum fixture (not shown) such that apressure differential can form between the sample in the treatmentvessel and the supplied vacuum. Once the vacuum is applied to thefixture, the pressure differential across the upper member 530 willcause the upper member 530 of the treatment vessel 508 to rupture andcause the lower member 550 to collapse into the funnel 592. The lowermember 550 should have sufficient strength so that it does not rupturewhere it bridges the opening in the small end of the funnel 592. Thepressure differential will cause the solid contents 596 of the treatmentvessel to be squeezed between the flexible lower member 550 of thetreatment vessel 508 and the relatively rigid funnel 592. This causesfluid 542 to be expelled from the solid materials 596 and collected inthe post-treatment vessel 598.

[0135] In certain other embodiments, a treatment vessel can be anampoule, vial, pouch, bag, or envelope. These and other treatmentvessels can be formed from such materials as polyethylene,polypropylene, poly(ethylene teraphthalate) (PET), polystyrene, acetal,silicone, polyvinyl chloride (PVC), phenolic, glasses and otherinorganic materials, metals such as aluminum and magnesium, andlaminates such as polyethylene/aluminum and polyethylene/polyester.Also, certain embodiments of a treatment vessel can be made by vacuumforming, injection molding, casting, and other thermal and non-thermalprocesses. In embodiments where samples flow through the sonic energy,capillary tubes, etched channels, and conduits may be the sample holderduring treatment as the sample flows through a structure. Additionally,free-falling drops, streams, non-moving free volumes, such as those ingravity less than one g, or a layer in a density gradient can be treateddirectly.

[0136] II. Materials for Treatment

[0137] A. Biological Materials

[0138] Many biological materials can be treated according the presentinvention. For example, such materials for treatment include, withoutlimitation, growing plant tissue such as root tips, meristem, andcallus, bone, yeast and other microorganisms with tough cell walls,bacterial cells and/or cultures on agar plates or in growth media, stemor blood cells, hybridomas and other cells from immortalized cell lines,and embryos. Additionally, other biological materials, such as serum andprotein preparations, can be treated with the processes of theinvention, including sterilization.

[0139] B. Binding Materials

[0140] Many binding reactions can be enhanced with treatments accordingto the invention. Binding reactions involve binding together two or moremolecules, for example, two nucleic acid molecules, by hybridization orother non-covalent binding. Binding reactions are found, for example, inan assay to detect binding, such as a specific staining reaction, in areaction such as the polymerase chain reaction where one nucleotidemolecule is a primer and the other is a substrate molecule to bereplicated, or in a binding interaction involving an antibody and themolecule it binds, such as an immunoassay. Reactions also can involvebinding of a substrate and a ligand. For example, a substrate such as anantibody or receptor can be immobilized on a support surface, for use inpurification or separation techniques of epitopes, ligands, and othermolecules.

[0141] C. Chemical and Mineral Materials

[0142] Organic and inorganic materials can be treated with controlledacoustic pulses according to the methods of the invention. The sonicpulses may be used to commute a solid material, particularly under afeedback control regime, or in arrays of multiple samples. As withbiological samples, individual organic and inorganic samples in an arraycan be treated in substantial isolation from the laboratory environment.Beside altering their physical integrity, materials can be dissolved insolvent fluids, such as liquids and gasses, or extracted with solvents.For example, dissolution of polymers in solvents can be very slowwithout stirring, but stirring multiple samples with current methods isdifficult and raises the possibility of cross-contamination betweensamples. However, stirring of multiple samples withoutcross-contamination between samples can be accomplished with apparatusand methods of the present invention

[0143] III. Treatment Applications

[0144] A. Altering Cell Accessibility

[0145] Sonicators can disrupt cells using frequencies around 20 kHz. Itis generally thought there are two ways in which ultrasound can affectcells, namely by heating and by cavitation, which is the interaction ofthe sound wave with small gas bubbles in the sample. Heating occursprimarily due to absorption of the sound energy by the medium or by thecontainer. For dilute aqueous systems, it is absorption by the containerthat is a main source of the heating. Heating is not desirable in sometreatment applications, as described herein. The heating associated withthe compression and cooling associated with the rarefaction of a soundwave is relatively small, even for intense sound.

[0146] According to the invention, controlled sonic pulses in a mediumare used to treat a sample containing biological material. The pulsescan be specifically adapted to preferentially interact with supportingmatrices in a biological material, such as plant cell walls orextracellular matrices such as bone or collagen, thereby lessening orremoving a barrier function of such matrices and facilitating theinsertion of extracellular components into a cell. In this application,the cell is minimally altered and cell viability is preserved. Thesepulses can be caused by shock waves or by sound waves. The waves can becreated external to the sample, or directly in the sample, via appliedmechanical devices. In experiments where thermal effects are negligible,there typically is no lysis, unless cavitation is present. Other modesof sonic energy can have different effects than disrupting a matrix andcan be used either with pre-treatment, with disrupting sonic energy, orby themselves. For, example the conditions to disrupt a matrix can bedifferent from those to permeabilize a cell membrane.

[0147] There are many possible mechanisms by which cavitation may affectcells and there is no consensus as to which mechanisms, if any,dominate. The principle mechanisms are thought to include shear,microjets, shock waves, sonochemistry, and other mechanisms, asdiscussed more fully below.

[0148] Shear: Significant shear forces are associated with the violentcollapse of bubbles. Because cell membranes are sensitive to shear, itis thought that cavitation may permeabilize cell membranes. In somecases, the membrane is apparently permeable for only a short time,during which molecules may be passed into or out of the cell. In othercases the cell may be lysed.

[0149] Microjets: Bubbles undergoing a violent collapse, particularlynear a boundary, such as a container wall, typically collapseasymmetrically and generate a liquid jet of fluid that passes throughthe bubble and into the boundary. The speed of this jet has beenmeasured to be hundreds of meters a second and is of great destructivepower. It may play a major role in the destruction of kidney stones byacoustic shock waves and may be a possible way of destroying bloodclots.

[0150] Shock wave: Collapse of a bubble spherically can generate anintense shock wave. This pressure can be thousands of atmospheres in theneighborhood of the bubble. The compressive stress of the shock wave maybe strong enough to cause cellular material to fail.

[0151] Sonochemistry: The pressure and temperatures in the bubble duringan inertial collapse can be extraordinarily high. In extreme examples,the gas can be excited sufficiently to produce light, termedsonoluminescence. Although the volume is small and the time durationshort, this phenomenon has been exploited to enhance chemical reactionrates. The production of free-radicals and other sonochemicals may alsoaffect cells.

[0152] Other: Other factors also may be involved. Vessel walls maycontribute cavitation nuclei. A plastic vessel with an aqueous fluid mayresult in a standing wave field due to internal reflections, as a resultof impedance mismatches between the fluid and the vessel walls. Examplesof sonolucent materials are thin latex and dialysis tubing. Tuberotation studies performed on continuous wave dosage with unfocusedultrasonics indicate that rotation has a significant effect onhemolysis. When cell contents were mechanically stirred duringinsonation, the cell lysis increased. These effects may be due toviscosity gradients set-up within the unfocused ultrasound field thatblock energy transmission.

[0153] Cellular lysis also can be aided by the addition of ultrasoundcontrast agents, such as air-based contrast agents orperfluorocarbon-based contrast agents. An example of an air-basedcontrast agent is a denatured albumin shell with air such as Albunex,available from Mallinckrodt, St. Louis, Mo., and an example of aperfluorocarbon-based contrast agent is a phospholipid coating withperfluoropropane gas such as MRX-130, available from ImaRxPharmaceutical Corp., Tucson, Ariz.

[0154] Air bubbles can block or reflect energy transmission. Interfacesbetween air and water result in efficient reflection of an incidentultrasound field.

[0155] The treatment dose is a complex waveform. Sections, orcomponents, of the waveforms can have different functions. For example,the waveform can have three components involved with sample mixing,sample lysis/disruption, and sample cooling.

[0156] In other current methods, sonolytic yield activity decreases withincreasing cell concentrations in in vitro systems that are treated withcontinuous ultrasound waves. In contrast, methods according to thepresent invention disrupt tissue structures with a complex waveform ofhigh intensity focused ultrasound, to avoid this problem.

[0157] Mixing can be an important, because it allows bubbles that mayhave been driven by radiation forces to the edges of the vessel chamberto be-brought into contact with the cell or tissue membranes. Thismixing promotes inertial, transient acoustic cavitation near the cellwalls, resulting in cellular lysis.

[0158] The acoustic dosage received by a sample can be likened to aradiation dosage received by a sample. In each case, a cumulative effectof the absorbed energy dose is observed. A computer-controlledpositioning system can control the cumulative energy dosage that eachsample receives. For example, a software program in the computer canactively control the cumulative energy dosage by treating the sampleuntil the system reaches a particular set-point, pausing energyapplication or otherwise allowing the sample to reequilibrate, andreinitiating energy application to allow a sample to receive a highercumulative dose while maintaining semi-isothermal conditions, such as a1 to 2 degree Centigrade temperature rise during exposure, than wouldotherwise be possible by continuous sonic energy application. This typeof system enables high energy to be introduced into a sample whilemaintaining thermal control of the process.

[0159] B. Extracting

[0160] In a variation of the method to alter cellular accessibilitydescribed above, controlled pulses in a medium can be used to treat asample containing biological material to extract a fraction or fractionsof the biological material. The pulses are specifically adapted topreferentially interact with supporting matrices, such as plant cellwalls or extracellular matrices such as bone or collagen, or materialshaving differences in rigidity or permeability in a biological material,thereby lessening or removing a barrier function of such matrices ormaterials. These pulses can be caused by shock waves or by sound waves.The waves can be created external to the sample, or directly in thesample, via applied mechanical means.

[0161] Using sound energy, as opposed to laser or other light energy todisrupt a biological object, can be useful. Sound is a directfluctuation of pressure on the sample. Pressure is a physical quantityand the measure of uniform stress defined as the force per unit area.The stress acting on a material induces strain which changes dimensionsof the material. The two main types of stress are a direct tensile orcompressive stress and shear stress. In general, the more brittle thematerial, the greater the disruptive effect of an abrupt, local increaseof otherwise uniform stress. Such a local stress can be created by somegeometric change at a surface or within the body of the sample. Forexample, biological tissue frozen at −70° C. may be more prone to stressfracture than at 4° C. In addition, a sharper change in geometric ormaterial properties tends to cause a greater stress concentration, whichin turn can yield a greater disruption. Sound waves may be focused. Incontrast, the energy transferred from a light source such as a laser toa sample is electromagnetic radiation that induces non-ionizingmolecular vibrations and breaks chemical bonds by ionizing. Mechanicalstress on objects larger than molecules generally cannot be readilycaused by electromagnetic waves, except via destructive local heating.

[0162] The supporting matrix of a biological sample can be disruptedwithout disrupting one or more selected internal structures of the cellscontained within the matrix. Representative examples of such samplesare: i) bone, in which a rigid matrix contains living cells of interest;ii) mammalian tissue samples, which contain living cells embedded in amatrix of elastic connective tissue and “glycocalyx” or intercellularmatrix; and iii) plant tissues, such as leaves, which contain cells in amatrix of cellulose, often crosslinked with other materials, of moderaterigidity. Virtually all living cells are gelatinous in texture, and canbe deformed to some extent without rupture or internal damage. Matrices,in contrast, are designed to support and protect cells, as well as toachieve other biological functions. In the three examples above, thematrices of bone and leaves are designed to provide rigidity to thestructure, while the support of most collagenous matrices has a stronglyelastic character. Thus, different protocols for example, amplitude,duration, number of pulses, and temperature of sample, may be used todisrupt different matrices by mechanical means without damaging thecellular material.

[0163] A bony matrix is both more rigid and denser than the cells itcontains. Bone is vulnerable to shock waves, both because the calcifiedmatrix will absorb the waves more efficiently than will the cells, andbecause the calcified matrix is weak under extensional strain, andthereby can fragment at stresses which will not damage the softer cells.Similar considerations apply to leaf matrix, although the contrast indensity and modulus is less. In either case, a pulse, preferably a shockwave, is applied at an amplitude which is sufficient to fatigue thematrix components while remaining below the amplitude required to damagethe cells. This intensity is determined readily for a particular type ofsample by minimal routine experimentation. In such experiments, theamplitude of each pulse applied to the sample, singly or in a train ofpulses, is varied to obtain the maximum rate of degradation of thematrix consistent with retention of the viability of the cells withinthe matrix. These parameters can be measured readily. For example,matrix degradation can be measured by variation in the compressivemodulus of the sample, while cell integrity is measured by dye exclusionfrom cells extracted from the matrix, such as, for bone,demineralization and treatment with collagenase. In the case of a moreelastic tissue, such as connective tissue, which is cross-linked but hasa high extension to break, the pulses are selected to excitepreferentially vibrational modes in the matrix in contrast to the cells.This can be done by selecting one or more frequencies of sound waves atwhich the relative absorptiveness of the matrix and the cells aremaximally different. Such frequencies are determined readily by routineexperimentation. A sequence of pulses may be required to differentiallyfatigue the matrix. The length of the pulses and the interval betweenthem are adjusted so that the degree of heating of the sample does notcause loss of integrity of the cells, and particularly of the criticalcomponents which are to be isolated.

[0164] Three areas to optimize for extraction are treatment waveform,mixing waveform, and positioning or dithering. One method to determinethe appropriate treatment and positioning parameters for a target samplefor extraction purposes is described below.

[0165] First, a solid sample is placed in a volume of liquid in about a1:1 ratio (weight/volume), in a treatment vessel. For example, 0.25 mlof methanol is added to 0.25 gm of leaf tissue in a 0.5 ml treatmentvessel. A single sample is placed within the focal zone of the sonicapparatus. Without using the treatment protocol, the mixing waveform isadjusted to provide “stirring” of the sample at the lowest amplitude,fewest cycles/burst, and lowest duty cycle. After the mixing waveformprotocol is defined, the disruption treatment waveform is adjusted byimmobilizing the target sample in the focal zone such that there is nomixing and no sample movement, such as dithering. Using a sonic energysource such as a piezoelectric transducer, the sample is subjected to aminimum number of cycles per burst, for example, three. For extractionpurposes, the amplitude is initially used with a nominal 500 mV setting.A portion of the sample is treated and inspected under a microscope forsigns of membrane disruption. Such inspection can be done in conjunctionwith dyes that stain intracellular organelles. The number ofcycles/burst is then increased until a particular desired tissuedisruption level is achieved in the immobilized portion of tissue. Witha fresh sample, and with a 1:1 ratio of tissue to liquid, thetemperature of the sample is monitored during a million cycle totaltreatment with an infra-red sensor directed to the top of a thinpolyethylene film covering the sample vessel. The duty cycle is adjustedto keep the temperature within predefined ranges, such as 4° C. within+/−2° C.

[0166] Once these treatment parameters are discerned for a particularsample, a control unit can be programmed with these data in order tocontrol treatment of other samples of the same or similar biologicaltype. Alternatively, such information can preprogrammed in the controlunit, and an apparatus user, through a user input interface, candesignate the biological material type to be treated such that thecontroller then runs through the predetermined treatment cycle. Otherinformation can be empirically determined for optimal treatment of aparticular biological material in a manner similar to that describedabove. For example, parameters such as treatment waveforms, mixingwaveforms, and sample positioning can be discerned. These parameters canvary depending upon the particular biological material, the particularliquid that surrounds the sample, and/or the particular treatment vesselused during treatment.

[0167] C. Introducing a Molecule into or Removing a Molecule from a Cell

[0168] Once a sample having a matrix has been sufficiently weakened orattenuated, but not to the point where a substantial number of cellscontained within the matrix are killed or lysed, an exposed target cellor cells become amenable to insertion of exogenous molecules bytechniques such as transfection or transformation. With some matrices,it may be convenient to isolate the cells from the matrices and then totransfect the cells. In other cases, it will be preferable, particularlyin an automated system, to perform the transfection directly on thetreated tissue sample, using solutions and conditions adapted from knowntechniques. Alternatively, in situations where a cell to be treated isnot situated within a matrix, the cell can be directly treated accordingto the process below without having to pre-treat the matrix. While thetreatment below is described mainly for transfection, methods andapparatus according to the present invention are equally applicable to atransformation process or other processes to introduce an exogenousmaterial into a permeabilized cell membrane.

[0169] In general, cool temperatures, less than 25° C., preferably lessthan 15° C., more preferably 4° C. or below, tend to minimize thedegradative effects of enzymes in the sample and thereby tend topreserve the integrity of biological components to be isolated. However,cells, especially mammalian cells, may maintain their viability betterat higher temperatures, such as 30 to 37° C. These temperatures alsoallow enzymes to be added to aid in the selective destruction of thematrix.

[0170] Alternatively, the sample temperature may be below 0° C. Exceptunder special conditions, this will freeze the sample, or maintain it ina frozen state. Freezing can be advantageous if it enhances thedisruption of the matrix while allowing the cell to remain relativelyintact. For example, ice crystals formed on freezing can be selectivelylarger outside of cells. Since such crystals may tend to absorbacoustical energy better than water, destruction of the matrix may beenhanced. While decreasing cell viability and integrity, such aprocedure could enhance the ease of transfection with exogenous materialafter thawing of the sample.

[0171] The waveforms used to alter the permeability of a cell arerefined depending on the particular application. Typically, the shockwave is characterized by a rapid shock front with a positive peakpressure, for example about 100 MPa, and a negative peak pressure, forexample about negative 10 MPa. This waveform is of a few microsecondduration, on the order of about 5 microseconds. If the negative peak isgreater than about 1 MPa, cavitation bubbles may form. Cavitation bubbleformation is also dependent upon the surrounding medium. For example,glycerol is a cavitation inhibitive medium; whereas, liquid water is acavitation promotive medium. The collapse of cavitation bubbles forms“microjets” and turbulence that impinge on the surrounding material.

[0172] Sound waves, namely acoustic waves at intensities below the shockthreshold, provide an alternative means of disrupting the matrix toallow access to the plasma membranes of the cells to allowtransformation. Such sound waves can be generated by any known process.As biological material is subjected to subzero temperatures, for exampleabout negative 5° C., most but not all of the water is in the solidphase. However, in certain biological tissues micro-domains of liquidwater still remain for several reasons, such as natural “antifreeze”molecules or regions of higher salt concentration. Therefore, as asample temperature is varied during the treatment with sound or shockwaves, microdomains of liquid water are able to form shock waves andinduce cavitation bubble formation and collapse, with the resultantshear stresses that impinge on surrounding tissues. Indeed, gradualalteration of the sample temperature can be desirable, as it providesfocused domains of liquid water for impingement on the surroundingmaterial. The waves can be applied to the samples either directly, aspiezoelectric pulses, or via an intervening medium. This medium may bewater, buffer, stabilizing medium for the target material to beisolated, or extraction medium for the target. An intervening mediumalso can be a solid, formed of a material which is intrinsically solid,or of a frozen solution. Waves also can be applied through a container,such as a microtiter plate.

[0173] The techniques useful for disrupting matrix structure can beadapted, and the improved technique can be used, to facilitate theincorporation of exogenous material into cells. The exogenous materialmay be DNA, RNA, other nucleic acid constructs, nucleic acid monomers,plasmids, vectors, viruses, saccharides, polysaccharides, amino acids,amino acid chains, enzymes, polymers, organic molecules, inorganicmolecules, proteins, cofactors, and/or visualization reagents such asfluorescent probes. In this application, shock waves or sonic waves areused to loosen the matrix, essentially as described above. However, theintensity of application of acoustic energy is kept sufficiently short,or below a critical energy threshold, so that cell integrity iscompletely maintained, as verified by a method such as dye exclusion.

[0174] At that point, or, optionally, previously, a solution orsuspension containing the material to be incorporated into the cells isadded to the sample. In one embodiment, the exogenous material isincorporated into the cells in a conventional manner, as is known in theart for cells with exposed plasma membranes. In another embodiment,acoustic energy is used to transiently permeabilize a plasma membrane tofacilitate introduction of exogenous materials into the cells. Theexogenous material may be present in the sample during the weakening ofthe matrix by acoustic energy. Even when the cells remain intact, asdetermined by dye exclusion or other viability measurements, the processof weakening the cell matrix by acoustic energy transiently destabilizesthe plasma membranes, increasing the uptake of exogenous macromoleculesand structures. If a further increase in the rate of incorporation isneeded, then the intensity or time of application of acoustic energy isslightly increased until the cell membrane becomes transientlypermettble. For example, a gentle pulse or wave is applied to themixture, with a predetermined amplitude. This amplitude can bedetermined readily in separate experiments on samples of the same typeto transiently make a plasma membrane of a cell type porous, in asimilar empirical manner to the steps described above for determining anappropriate treatment to disrupt a matrix. During the transient porousstate, exogenous materials diffuse into the cell and the materials aretrapped there once the sonic or shock pulse is removed.

[0175] A major advantage of these methods for transfection, or otherincorporation of exogenous material into living cells, is that themethods are readily amenable to scale-up, to automation, and to markedreduction in sample size and reagent volume. The wells of microplatescan be used for sonic treatment, transfection, and post-transfectiondemonstration of successful incorporation of the added material. Forexample, extracellular non-incorporated reagent, for example afluorescent material, can be inactivated by a material that does notpass the cell membrane, such as an enzyme, or certain hydrophilic oramphiphilic small-molecule reagents. Then the presence or absence of therequired material can be determined directly in the sample, for exampleby spectroscopy. Thus, the methods are adaptable to large scaleautomation, in large part because they do not require the isolation ofthe cells from their matrix. Additionally, these methods are amenable toa continuous flow process such as that described for sterilization,below. For example, the sonic energy treatment can be different forpermeabilization than for sterilization, but the sample to be treatedcan be flowed through an apparatus similar to that described forsterilization in FIG. 7.

[0176] The permeabilized cells can be transformed or transfected, usingtechniques known to those skilled in the art, for example,electroporation, vacuum transfection, or using viral vectors,agrobacterium, liposomes or other delivery vehicles, plasmids, or nakednucleic acids. The buffer conditions may be altered during the process.For example, the initial permeabilization may occur with chemicals toselectively alter the external cell wall, while during the nuclear wallpermeabilization step, other chemicals or biochemicals may be added toprompt selective uptake.

[0177] Additionally, with the process of permeabilization and with themixing profile, other techniques of gene transfer may be augmented.Examples include, calcium phosphate coprecipitation, electroporation,and receptor-dependent processes.

[0178] D. Sterilizing

[0179] The terms “sterilize,” “disinfect,” “preserve,” decontaminate,”“inactivation,” “disinfect,” and “kill” are used interchangeably herein,unless otherwise demanded by the context. “Sterilization,” namelykilling of all organisms, may not be synonymous in certain operationswith “decontamination,” for example, when the contaminant is non-living,such as a protein or prion. These terms, typically, mean the substantialelimination of or interference with any activity of a particularorganism and/or particle.

[0180] Methods for permeabilization and extraction, described above, canbe modified to sterilize a sample. The apparatus and methods forsterilizing can be optimized for efficient sterilization of particularmaterials in particular volumes and containers. For a particularmaterial to be sterilized, an initial set of conditions is selected.Such conditions can include selection of a type of sonic pulsegenerator, intensity of sonic energy, frequency of sonic energy, whererelevant, and/or like variables. The conditions also can include volume,mode of transport, and/or exposure of the materials to be sterilized.Then, the initial conditions and near variants are applied to thesample, and the percentage of cells or viruses killed is determined bystandard assay conditions. Further variables are selected for change.Accordingly, a zone of maximal killing of the test organism is found.Finally, other variables, such as flow rate and/or length and/orintensity of sonic exposure, are optimized to provide both a technicalsolution and a commercially useful solution to the problem ofsterilizing a particular material. Any of these empirically determinedvalues can be programmed into a control system of an apparatus used forsterilization to actively control sterilization, or the apparatus canhave these values previously determined such that a user need onlyselect a predetermined sterilization mode an the apparatus.

[0181] For many liquids, adequate sterilization is provided bydestroying the cell walls of bacteria, fungi, and other living cells.This result is accomplished by using frequencies and wavelengths ofsound which preferentially excite the membranes of the cells whileminimally heating the solution until the cells are lysed. In mostcellular organisms, opening the membrane and allowing the contents tomix with an extracellular fluid will kill the organism.

[0182] Viruses can be opened to the solution by similar processing. Inthe case of viruses, exposure of their internal nucleic acid to thesolution may not be adequate to completely inactivate them, since thenaked DNA or RNA can also be infectious. Adjuncts such as iodine ornucleic-acid digesting enzymes in the solution can be provided tocomplete the inactivation of the viruses.

[0183] Now, referring to FIG. 7, a schematic illustration for anapparatus 50 to sterilize a continuous flow fluid is shown. For example,but without limitation, the apparatus can be used to sterilize blood orother fluids supplied to a patient. In this embodiment, fluid flowsthrough the lumen of a conduit 54 between a first connector 62 and asecond connector 56. The connectors 56, 62 can be Luer fittings and theconnectors can be connected with other tubing and/or devices (not shown)that provide or receive the fluid. The fluid moves between theconnectors 56, 62 in a direction indicated by an arrow 58. A sonicenergy source 60, such as a high intensity focused ultrasoundtransducer, is located adjacent the conduit 54 and the sonic energy isemitted to a focal zone at least partially within the conduit 54. Manydifferent arrangements of a sonic energy source or sources are possible,such that sonic energy is emitted to a focal zone into the fluidcontained within the conduit. The temperature of the fluid flowingthrough the conduit 54 can be monitored with a sensor (not shown) that,for example, receives infrared energy from the fluid as it flows throughthe conduit 54. Alternatively, the conduit can have at least one windowor thin membrane portion which allows infrared radiation to pass throughto the sensor. A computer with an adaptive control can provide preciseand accurate control of the temperature of the medical fluid during thetreatment in a manner similar to that described above. Also, during theultrasonic treatment, feedback control can stabilize the temperature ata desired value, in a manner similar to that described above, tomaintain the integrity and/or viability of fragile components within thefluid. For example, if the fluid is blood, one fragile component that ismaintained can be Factor VIII. In addition, flowing the fluid past thefocal point maintains a “bubble-free” focal zone. While blood might beremoved from a patient, treated according to the invention outside thepatient, that is treated ex vivo, and returned to the patient, othertreatment situations are possible. For example, one person's blood canbe removed and treated according to the invention and then given to asecond person during a transfusion. Additionally, the sterilizingqualities of treatments according to the invention are contemplated tobe useful whenever a fluid needs to be sterilized.

[0184] In another sterilizing treatment mode and apparatus, andespecially for high-volume applications, a wide, shallow zone ofsterilizing sonic energy can be created by apposition of a pair ofplates to form a sterilizing cell. At least one of the plates is anemitter of sonic energy. The sterilizing cell is sealed appropriatelysuch that the cell has a sealed inner volume, with connections for fluidflow into and out of the cell. Fluid flow through the cell can besubstantially laminar under these circumstances, facilitating properflow rate selection to provide sufficient exposure of the fluid to thesonic energy to produce sterilization.

[0185] In an alternative sterilizing treatment mode and apparatus, fluidis conveyed through a zone of sterilizing sonic energy by being pumpedthrough the zone in a conduit. The conduit itself may be immersed in aliquid or solid material that is designed to improve the efficiency withwhich sonic energy from the sonic energy emitter is provided to theconveyed fluid. The conduit also can be directly connected to a sourceof sonic energy, such as a tubular piezoelectric wave source. Ifchemical compatibility is adequate, a portion of the conduit itself maybe made of a material that can generate the sonic waves, such as apiezoelectric ceramic. Alternatively, any of these treatment processesmay be a manufacturing batch process for intravenous products.

[0186] E. Mixing, Stirring, and Heating

[0187] In fluid samples, including powdered and granular media andgasses, sample mixing is conventionally performed by vortexing orstirring, or other methods such as inversion of a sample containing anair space, and shaking. Vortexing is essentially achieved by mechanicalmotion of the entire vessel while stirring involves mechanical contactof a driven device with a fluid. Stirring is accomplished with a varietyof devices, for example with propellers, impellers, paddles, andmagnetic stir bars. One problem with these methods is that it isdifficult to increase their scale in order to handle dozens or hundredsof sample vessels at once. Another problem with these methods is thedifficulty of mixing multiple samples while keeping the each samplesubstantially free from contamination. As described in more detailbelow, methods according to the invention can use sonic energy to mix asample while avoiding problems with contamination. Factors, such asfocusing the sonic energy, as well as otherwise controlling an acousticwaveform of the sonic energy, can be used to selectively mix a sample,for example, through acoustic streaming and/or microstreaming.

[0188] A fluid sample can be mixed controllably using the systemdescribed herein. No direct contact between the material to be mixed andthe sonic energy source is required. When the material to be mixed is ina treatment vessel, such as a microplate, the treatment vessel itself isnot necessarily touched by the source and is typically coupled to thesource by a fluid bath.

[0189] In certain embodiments, a treatment process for sample mixing ina treatment vessel can be summarized as follows. First, a sample istreated with sonic energy at a relatively high first treatment power inorder to heat the sample by absorption of acoustic energy. Second, thesample is mixed at a second sonic energy power, which may be the same orlower than the first treatment power, to cool the sample back to itsoriginal temperature by forcing convection through material in thetreatment vessel, which can be in contact with a fixed-temperature bathor reservoir.

[0190] In some embodiments, a source of focused ultrasonic waves isused. The source is mounted in a water bath or equivalent, which canprovide temperature control. The microplate, with samples in the wells,is positioned so that the focus of the beam is within the well. Theplate is positioned so that the bottoms of the wells are in contact withor immersed in the water or other fluid in the bath. Then, a burst ofultrasonic energy is applied to the well. This burst will cause stirringin the well, by formation of a convection cell. The stirring is easilyvisualized by adding particulate material to the wells, or by adding adye in a denser or lighter solution.

[0191] It is possible to select a sound field which will stir all of thewells of a plate at one time. In one embodiment, a substantially uniformfield is projected to the plate by a source, which preferentiallyexcites the bottoms of the wells. This excitation in turns drivesconvective flow in each of the wells.

[0192] In any embodiment, it can be useful to move the sample treatmentvessel, such as by “dithering” the plate or well being treated relativeto the source. Dithering, as used in optics and in laser printing, is arapid side to side two or three dimensional movement of the energysource and/or the target. Dithering, or other types of motion, can evenout variations in source intensity due to variations in the emittedsonic energy or the location of the sample with respect to the source.Dithering can also prevent particulates from accumulating at the wall ofthe well.

[0193] F. Enhancing Reactions and Separations

[0194] In certain embodiments, temperature, mixing, or both can becontrolled with ultrasonic energy to enhance a chemical reaction. Forexample, the association rate between a ligand present in a sample to betreated and an exogenously supplied binding partner can be accelerated.In another example, an assay is performed where temperature ismaintained and mixing is increased to improve association of two or moremolecules compared to ambient conditions. It is possible to combine thevarious aspects of the process described herein by first subjecting amixture to heat and mixing in order to separate a ligand or analyte inthe mixture from endogenous binding partners in the mixture. Thetemperature, mixing, or both, are changed from the initial condition toenhance ligand complex formation with an exogenously supplied bindingpartner relative to ligand/endogenous binding partner complex formationat ambient temperature and mixing. Generally, the second temperatureand/or mixing conditions are intermediate between ambient conditions andthe conditions used in the first separating step above. At the secondtemperature and mixing condition, the separated ligand is reacted withthe exogenously supplied binding partner.

[0195] Polymerase Chain Reaction (“PCR”) Thermal Cycling

[0196] One of the bottlenecks of the PCR technique is cooling time. Theheating cycle is rapid; however, cooling is limited by convection. Evenin biochip formats, in which DNA or another target molecule isimmobilized in an array on a microdevice, there is no “active” coolingprocess. However, certain embodiments of the invention can be used toovercome this bottleneck.

[0197] In certain embodiments, a treatment process can be used to bothheat and cool the sample rapidly with little overshoot from a baselinetemperature at which the primer and target to be amplified anneal. Theprocess can be summarized as follows. A sample is treated withrelatively high power sonic energy such that the sample absorbs sonicenergy and is heated. Then, the sample is mixed at low power to cool thesample by forcing convection, which may be accomplished in conjunctionwith a cool water bath. In some embodiments of the apparatus, the systemis a “dry top” system, that is, a system in which a microplate,typically with its top temporarily sealed with plastic film, floats onor is partially immersed in a controlled-temperature bath. In thisarrangement, the PCR reaction may be monitored in real-time fortemperature using, for example, an infra-red detection probe, and forreaction products by examining the incorporation of fluorescent dyetagged nucleic acid probes into the PCR product. This “dry top” systempermits real-time analysis and control of the process. Information fromthe temperature sensor can be used in a feedback loop to control theduty cycle of the acoustic input, such as the number of bursts/second,or otherwise control the amount of heating. Also, fluorescence from anintercalated probe can provide a computer with information on whichwells have reached a certain point in the reaction, such as when aparticular level of fluorescence is sensed, allowing, for example, thecomputer to control application of sonic energy or sample location suchthat certain wells are skipped in the processing cycle until other wellshave attained the same point in the reaction or that certain wells arenot processed further.

[0198] G. Purification, Separation, and Reaction Control

[0199] Focused sonic fields can be used to enhance separations. As notedelsewhere, sonic foci can be used to diminish or eliminate wall effectsin fluid flow, which is an important element of many separationprocesses, such as chromatography including gas chromatography, sizeexclusion chromatography, ion exchange chromatography, and other knownforms, including filed-flow fractionation. The ability to remotelymodulate and/or reduce or eliminate the velocity and concentrationgradients of a flowing stream is applicable in a wide variety ofsituations.

[0200] Sonic fields also can be used to minimize concentrationpolarization in membrane processes, including particle classification,filtration of fine particles and colloids, ultrafiltration, reverseosmosis, and similar processes. Concentration polarization is the resultof the tendency of filtered material to be present at high concentrationin a layer on the filter. This layer has a low fluid concentration and,thus, diminishes the rate of filtration as the filtered solution becomesmore concentrated, or as the layer thickens. This layer can be stirredremotely by focused sonic energy of low to moderate intensity. Flowrate, thus, can be enhanced without significant cost in energy ormembrane life.

[0201] Such sonic energy fields can be used to enhance reaction rates ina viscous medium, by providing remote stirring on a micro scale withminimal heating and/or sample damage. For example, some assays rely onthe absorption of analytes by reagents, such as antibodies, which arebound to macroscopic particles. In a viscous fluid to be analyzed, suchas sputum or homogenized stool, the ability to stir such a sampleremotely, aseptically, and essentially isothermally can significantlydecrease the time required to obtain equilibrium of the analyte with thereagents on the particle.

[0202] Likewise, any bimolecular (second-order) reaction where thereactants are not mixed at a molecular scale, each homogenouslydissolved in the same phase, potentially can be accelerated by sonicstirring. At scales larger than a few nanometers, convection or stirringcan potentially minimize local concentration gradients and therebyincrease the rate of reaction. This effect can be important when bothreactants are macromolecules, such as an antibody and a large target forthe antibody, such as a cell, since their diffusion rates are relativelyslow and desorption rates may not be significant.

[0203] These advantages may be realized inexpensively on multiplesamples in an array, such as a microtiter plate. The use of remote sonicmixing provides a substantially instantaneous start time to a reactionwhen the sample and analytical reagents are of different densities,because in small vessels, such as the wells of a 96 or 384 well plate,little mixing will occur when a normal-density sample (about 1 g/cc) islayered over a higher-density reagent mixture. Remote sonic mixing canstart the reaction at a defined time and control its rate, whenrequired. The stepping and dithering functions allow multiple readingsof the progress of the reaction to be made. The mode of detectingreaction conditions can be varied between samples if necessary. In fact,observations by multiple monitoring techniques, such as the use ofdiffering optical techniques, can be used on the same sample at eachpass through one or more detection regions.

[0204] H. Further Uses for Remotely Actuated and Controlled SolutionMixing with Sonic Energy

[0205] Control of sonic energy emission, sonic energy characteristics,and/or location of a target relative to sonic energy also can be used topump and control the flow rate of liquids, especially in capillaries;enhance chemical reactions, such as enhancing second-order reactionrates; increase effective Reynolds number in fluid flow; and control thedispensing of semi-solid substances.

[0206] By focusing sonic energy and positioning it near a wall of avessel, a wall of a tube, or another discontinuity in a fluid path, manylocal differences in the distribution of materials within a sampleand/or spatially-derived reaction barriers, particularly in reactive andflowing systems, can be reduced to the minimum delays required formicroscopic diffusion. Put differently, enhanced mixing can be obtainedin situations where imperfect mixing is common. The range of thesesituations is illustrated below.

[0207] Control of Flow Rates of Fluids

[0208] Miniaturization of analytical methods, such as analysis on achip, require concomitantly miniature capillary-sized dimensions forfluid flow paths. Sonic excitation provides a convenient, simple, andsterile manner to accelerate flow in capillaries. During excitation, thefluid is locally turbulent, and so flows more readily. By selective ortimed local sonic excitation, optionally controlled with a feedbackloop, the rate of flow through complex microfluidic paths can beremotely manipulated in a controlled manner.

[0209] Increase of Effective Reynolds Number in Fluid Flow

[0210] At low Reynolds numbers, the velocity profile of laminar fluidflow in a pipe or other conduit is approximately parabolic. Fluid at thecenter of the pipe is flowing significantly faster than fluid near thewall. Therefore, conversion of fluid carried in the pipe from one fluidto another is quite slow, and, in principle, infinitely slow.

[0211] This effect effectively vanishes at higher Reynolds numbersbecause turbulence mixes the fluid at the center with fluid at theperiphery very rapidly, so that composition differences are rapidlyeliminated. However, there are significant disadvantages to operating afluid conduit under turbulent conditions, including high backpressureand correspondingly high energy expenditure.

[0212] If sonic energy is focussed in, on, or near the wall of the pipe,near the fluid/wall boundary, then local turbulence can be obtainedwithout a high rate of bulk fluid flow. Excitation of the near-wallfluid in a continuous, scanned, or burst mode can lead to rapidhomogenization of the fluid composition just downstream of the excitedzone. This will sharpen the front between any two fluids passing througha pipe in succession.

[0213] This effect is useful in several areas, including chromatography;fluid flow in analytical devices, such as clinical chemistry analyzers;and conversion of the fluid in a pipeline from one grade or type toanother. Since most of the effect occurs in a narrow zone, only a narrowzone of the conduit typically needs to be sonically excited. Forexample, in some applications, the focal zone of the sonic energy can bethe region closest to a valve or other device which initiates the switchof composition. In any of these applications, feedback control can bebased on local temperature rise in the fluid at a point near to ordownstream of the excitation region.

[0214] Enhancement of Second-Order Reaction Rates

[0215] Microsonication can be used to speed up, or to homogenize, therate of chemical reactions in a viscous medium. The flow of individualmolecules, and of heat, is generally slower in a more viscous medium.For example, it is more difficult to mix molasses with water than to mixvinegar with water. Similarly, in an aqueous solution, it becomesincreasingly difficult to maintain the rate at which soluble monomersundergo a polymerization reaction, forming a soluble polymer, as themolecular weight of the polymer increases with each added monomer,because the viscosity of the solution increases.

[0216] Mixing of molasses and water with a stirrer is simple, but noteasily sterile, and a polymer can be degraded by shear caused bystirring with a stirrer. Focussed sonication can readily mixpre-sterilized liquids in a remote manner without contamination. Focusedsonic energy also can mix polymerizing materials without application ofmacroscopic shear, and so can minimize shear degradation of the formedpolymer. Similarly, a polymerase chain reaction can be accelerated bybrief pulses of sonic energy, or by longer pulses which also provide thedesired temperature increases, to prevent the retardation of thereaction due to local depletion of the nucleotide triphosphate monomers.

[0217] Controlled Dispensing of Semi-Solid Substances

[0218] Highly viscous liquids, including materials which effectively actas solids or near-solids, can flow at an increased rate when sonicallyexcited by a remote or local sonic source. This excitation may be underfeedback control. This effect can be caused by local reduction ofimpedance to flow by walls of a conduit, as described above, and bylocal heating from sonic energy input. As a simple example, theeffective viscosity of an ink jet ink, and thus the rate of itsdelivery, can be controlled by focused, localized sonic energy delivery.Analogous uses are possible wherever the viscosity of a fluid, includinga semi-solid or a solid capable of melting, is significant. Likewise,flow of particulate materials in a fluid where the particles areinsoluble in the fluid can be selectively stimulated to occur, or beaccelerated, with focussed, controlled sonic waveforms.

[0219] Various embodiments of the present invention will be furtherunderstood by reference to the following non-limiting examples.

EXAMPLES Example 1 Isolation of Intracellular Components From Cells

[0220] To further aid in the understanding of this invention, aprocedure for the isolation of intracellular components from cellsimbedded in a matrix is described. A sample of about 100 cu. mm. volumeis placed into each well of a multiwell plate, such as a 96 well plate,having a capacity of about 200 microliters (200 cu. mm). The entireplate is then frozen and reduced to about minus 40° C. Then, about 100microliters of an extraction solution, precooled to 4° C., is added toeach well while the plate is held at minus 40° C. This maintains thesample temperature at minus 20° C. or less, while providing a smoothsurface in the well for coupling to the wave source. A sheet of flexibleplastic foil is optionally affixed to the plate to prevent crosscontamination between the contents of the wells, or between the wellsand the wave source. A piezoelectric wave source is provided andpositioned on the plate. The source has 96 pins in the appropriatearray, and each pin is connected, preferably removably, to apiezoelectric driver carried in a common holder for the pins, thedrivers,.and associated circuitry. Then, a series of electrical pulsesis applied to the drivers to generate shock waves in the samples. Theapplication of the series is preferably driven by an automatedcontroller, such as a custom chip or a programmed computer. The wavesource is removed, preferably robotically, and the plate is rapidlywarmed to 4° C. The 96 solutions in the wells of the plate are agitatedby a mild sonic vibration, at an intensity too low to mechanicallydamage the target molecules. After a defined incubation period, such as30 seconds, the plate, still bearing the plastic foil, is removed to acentrifuge to pellet debris, and the top 50 microliters of each sampleis removed for further analysis.

Example 2 Steady-State Temperature Control with Controlled WaveformGeneration to Uniformly Mix a Sample

[0221] In this example, the duty cycle of an ultrasound treatment wasvaried to reduce steady-state temperature rise within a sample, comparedto a continuous wave, 100% duty cycle.

[0222] A 1.1 MHz high power transducer was applied to a sample treatmentvessel. The sample treatment vessel was constructed of two layers ofthin film. The bottom consisted of a ⅜ inch (9.5 mm) diameterhemispherical “bubble” from bubble-wrap packing material with the flatside cut away to yield a ⅜ inch diameter hemisphere, made of a thinplastic. The top layer was 0.001 inch thick saran film again, a thinplastic. The vessel, having approximately a 300 μl total volume,contained a liquid sample of 50% methanolic solution, that is 1:1methanol volume to water volume. The sample treatment vessel wasinserted into a frame that allowed the bubble to protrude into the focalzone of the transducer in a water bath. The vessel was then placed intoa water bath at 3.5° C. The saran film top side wastexposed to the air.The temperature of the internal liquid was measured with a J-typethermocouple at the periphery of the treatment vessel and a thermocouplemeter, Omega model #DP116-JC2. An input signal of 500 mV sine waves at1.1 MHz frequency generated by an arbitrary waveform generator input toa 55 dB RF amplifier was applied to the transducer resulting in a peakpositive pressure of approximately 15 MPa and a peak negative pressureof approximately −6 MPa in the focal zone of the resulting acousticfield. The transducer was focused on the sample vessel containing themethanolic solution such that the sonic energy entered the vesselthrough the bottom film made from the bubble wrap and converged withinthe vessel. The acoustic dosage received by the sample was 1,000cycles/burst, 10,000 bursts per dose for a total dose of 10,000,000cycles. The same sample was treated with duty cycles of 1%, 5%, 10%, and20%.

[0223] A steady state condition was obtained after an initial transienttemperature change. In all cases the transient temperature changeoccurred within the first 30 seconds and the temperature became stablefor the remainder of the dose, up to several minutes, depending on theduty cycle. The data are presented in Table 1, below. TABLE 1Temperature rise in degrees Celsius as a function of duty cycle andamplitude. Temperature Rise Temperature Duty cycle at 500 mV Rise at 750mV 1% 0.6 1.1 5% 1.1 2.7 10% 2.0 4.9 20% 2.7 6.4

[0224] This example demonstrates that high intensity focused ultrasonicenergy can be focused on an in vitro sample without deleterious heatgeneration. The sample treatment vessel described here is optimized forefficient heat transfer and acoustic transparency. By providing areliable way to monitor the status of the sample, such as an infraredsensing temperature probe, and control over the electrical waveforminput to the ultrasound transducer, the ultrasound signal can beoptimized to maximize energy transfer while minimizing temperature riseor other deleterious effects.

Example 3 Increasing Extraction Output by Using Infrared TemperatureFeedback to Vary Either the Duty Cycle and/or the Voltage

[0225] A 1.1 MHz high power transducer was configured to treat a samplein a treatment vessel constructed in a manner as described in Example 2.The acoustic dosage received by a sample of leaf tissue suspended in amethanol solution in the vessel was 500 cycles/burst, 2,000 bursts perdose, with a variable duty cycle. The starting temperature of the vesselwas less than 1° C.

[0226] Upon initiation of the treatment, the temperature within thevessel stabilized within 10 seconds and remained stable during thedosage interval of up to ten minutes. The duty cycle was adjusted tocontrol the temperature rise. The effect of the dose was visuallysimilar, whether the dose was received by the sample as one long burstin a continuous wave (“CW”) or as an accumulation of shorter bursts,having a duty cycle less than 100%.

[0227] The results are shown graphically in FIG. 8. At a 500 mV waveamplitude, the temperature of the treated sample rose approximately 0.5°C., 1.9° C., 2.8° C., and 3.0° C. from a starting temperature ofapproximately 0.0° C. at duty cycles of 1%, 5%, 10%, and 20%,respectively. At a 750 mV wave amplitude, the temperature of the treatedsample rose approximately 1.1° C., 2.7° C., 4.9° C., and 6.4° C. from astarting temperature of approximately 0.0° C. at duty cycles of 1%, 5%,10%, and 20%, respectively. These data are useful for constructing asonic energy control system either with or without a feedback loop.

Example 4 Sample Mixing and Disruption with Synchronized Intra-SampleFocal Zone Positioning

[0228] This example indicates that movement of a sample through thesonic energy field has beneficial effects. When a focused ultrasonicdose with non-optimized mixing waveforms was applied to a leaf tissuesample that contained a heterogeneous mixture of leaf lamina, stalks,veins, and potting soil, a small portion of the sample was disrupted.With an ultrasonic dose and a stationary focal zone, the largerparticles of leaf clumps, debris, etc. were visibly “pushed” to theperipheral edges, while the smaller particles remained within or nearthe focal zone. When the focal zone was swept across the sample slowlyin a circular motion during the treatment, clumps of material around theperiphery of the treatment vessel were brought into the focal zone andwere visibly broken up. Manually moving the sample across the focal zonewith a Newport Series 462 x, y positioning stage resulted in betterresults than with a stationary focal zone. However, while manuallymoving the sample was beneficial for treatment purposes, the manualmoving results were not as precisely repeatable as they can be with acomputer-controlled positioning system such as was used in Example 5,below.

[0229] A benefit of automated movement of the sample relative to thetransducer, also known as x-y dithering, can be to prevent a bubbleshield from forming and blocking cavitation within the sample treatmentvessel. Another benefit is that the x-y dithering can also enhancetreatment of sample suspensions that have a high viscosity and do notmix well. Dithering becomes increasingly advantageous as the sampletreatment vessel becomes significantly larger than the focal zone.

[0230] The automated movement of the sample relative to the ultrasoundtreatment can be advantageous with an unfocused transducer, such as a 20kHz cell disrupter ultrasonic probe, because, for example, relativemotion between the sample and the ultrasound source during treatmentprevents the suspended particles in the sample from collectingpreferentially at the low intensity nodes of the acoustic field.

Example 5 Extraction of Amino Acids from Plant Leaf Tissue withUltrasound and Positional Dithering

[0231] Samples of approximately 100 mg of A. thaliana leaf tissue werecollected and flash frozen in liquid nitrogen. The frozen material wasstored in 2 ml vials at −75° C. until the day of the experiment. Thesamples were then transferred to dry ice and placed into individualvessels for treatment. The treatment vessels consisted of ⅜ inch (9.5mm) diameter hemispherical “bubbles” made from bubble-wrap packagingmaterial, as described in Example 2. The tissue samples were placed intothe sample treatment vessels with approximately 200 microliters ofpre-chilled, 4° C. 90% methanol:water (9:1, v/v). The samples were thenwarmed to approximately 4° C. for the treatment.

[0232] The apparatus contained a transducer in a water bath at roomtemperature, approximately 24° C., with the treatment vessel ultimatelypositioned in another inner water bath having an acousticallytransparent film located in the path of the converging acoustic waves.The inner water bath was chilled with copper coils to about 4-6° C. Thesample treatment vessels were inserted into a computer-controlled x, y,z positioning system. The samples were then aligned in the focal zone ofthe transducer, using predetermined positions, with the beginning of thefocal zone convergence at approximately 2 mm inside of the treatmentvessel.

[0233] Four conditions were tested during the experiment. First, anexperimental condition (type one) was tested, where a leaf tissue samplewas placed in a methanol solution, subjected to sonic energy,centrifuged, and the supernatant removed. This supernatant was testedfor the presence of amino acids, peptides, proteins, and other primaryamines (“amino acids”). The pellet was resuspended in methanol,vortexed, centrifuged, and the supernatant removed. This secondsupernatant was tested for the presence of amino acids. The amount ofamino acids recovered from the first “extraction” step was divided bythe total amino acids extracted during both extractions to determine thefraction of amino acids recovered during the first extraction of thetotal number of amino acids present. Second, an experimental condition(type two) was tested, similar to the first experimental condition,except that during sonic energy exposure, the sample was dithered, beingmoved relative to the sonic energy source.

[0234] The waveform used in all of the treatments in this example had anamplitude of 500 mV, a frequency of 1.1 MHz, bursts of 1000 cycles and aduty cycle of 10%. This waveform was previously found to effectivelytreat and mix the sample without generating excessive heat, as describedabove. All of the samples received 10,000 of these bursts.

[0235] The sample was continually mixed during the treatment. Thepre-defined treatment parameters insured that both the disruption phaseand mixing phase occurred at constant temperature. From otherexperiments, it is known that the temperature remained essentiallyconstant and was essentially isothermal with the inner bath. The innerbath temperature ranged from 4-6° C., and the sample temperature alsoranged from 4-6° C. during the process.

[0236] Immediately following the experimental treatment, as much of thesample as possible was transferred to 0.5 ml polypropylene vials. Thetypical recovery was greater than 75% of the liquid and solid sampletransferred. The samples were then gently vortexed for 10 seconds, andwere centrifuged for 2 minutes at 5,000 rpm with a smallmicrocentrifuge. The supernatant was immediately transferred to anothermicrocentrifuge tube and was placed in a −80° C. freezer until analysis.

[0237] There were two controls. First, a sham control was used that wasidentical to the first experimental condition, but without treatmentwith ultrasonic energy. The RF amplifier was not turned on. Second, amethanol double extraction control process, such as one that might beused for extraction without treating a sample with sonic energy, wasutilized to compare with the ultrasonic treatment process. The controlprocess was to add a 90% methanol solution to the plant tissue, let themixture stand at room temperature for 2 hours, vortex the mixture forone minute, centrifuge the mixture for 5 minutes, remove the supernatantin a first extraction and perform an amino acid assay. Methanolextraction was repeated on the pellet that remained after thesupernatant was removed during the first extraction step. Again, thesupernatants from the first and second extractions in all of thecontrols were tested for amino acid content and the amino acid contentwas expressed as a fraction of the total amount of amino acids collectedduring the first and second extractions, combined. All control samplesunderwent one freeze/thaw cycle, just as the two experimental conditionsdid, and, consequently, there was tissue lysis and amino acid releasedue to the mechanical freeze/thaw effects. Thus, this freeze/thaw effectis controlled for in the results.

[0238] Typically, in either of the two experimental conditions, about 10μmole of amino acids per wet gram of tissue was recovered from the firstextraction and about an additional 3-5 μmole of amino acids per wet gramweight of tissue was recovered from the second extraction. Allextractions were performed in duplicate or triplicate.

[0239] The extract samples, namely the supernatant removed during bothextractions during both of the experimental conditions and both of thecontrol conditions were assayed for total amino acids with afluorescamine assay. The fluorescamine reacts to form stable fluorescentsubstances with amino acids, peptides, proteins, and other primaryamines under mild reaction conditions. Fluorescamine (0.07 ml, 14.5 mM)was added to 0.08 ml of triethylamine/acetate (pH 8.6), and this mixturewas added to 20-50 μl aliquots of the extract sample and, subsequently,was brought up to a total volume of 200 μl with 100% methanol in amicrotiter plate. The mixture was incubated for 30 minutes at 25° C. Themicrotiter plate was then excited at 395 nm and the emission detected at460 nm in a standard fluorescent plate reader. Standard curves includeda mixture of phenylalanine, alanine, and leucine in 10% acetonitrile inthe range of 0.1 to 10 mM, such that the fluorescent signature of theextract samples could be compared with a standard to determine the molarconcentration of amino acids in the samples.

[0240] The results are expressed as micromole of amino acids per gram ofwet tissue for both first and second extractions. The effectiveness ofthe treatment is expressed as the first extraction as a percentage ofthe total extracted amino acids in both extractions.

[0241] The experimental results, are shown in Table 2, below, and theseresults were averaged for comparison, as shown in Table 3. The resultsindicate that: (1) the control process samples of fresh frozen tissueextracted with a 90% methanolic solution that were not treated withfocused sonic pulses, but were vortexed, centrifuged and assayed as forthe experimental samples, resulted in approximately 82% of the totalamino acids being present in the first extraction; (2) the sham-controlsamples which were inserted into the treatment vessel, incubated at 4°C. for eight minutes (equal to the longest treatment interval),vortexed, and centrifuged resulted in approximately 85% of the totalamino acids being in the first extraction; (3) experimental samples(type one) that were exposed to an ultrasonic dose of 500 mV, 1,000cycles/burst, 10,000 bursts, and a cumulative dose of 10 million cycles,without dithering, resulted in approximately 94% recovery of total aminoacids in the first extraction with a coefficient of variation of lessthan 2%; and (4) experimental samples (type two) that were treated witha similar dosage as with type one, with the addition of positionaldithering of the sample relative to the ultrasound source during thetreatment process, resulted in greater than 99% recovery of total aminoacids in the first extraction, with a less than 1% coefficient ofvariation. The experimental samples (type one and two) appeared greenand cloudy like “pea-soup” following treatment, whereas the shamcontrols were clear and tinged green with chlorophyll.

[0242] In the experimentally processed samples, stems did not appear tobe affected by the process; however, leafy tissue was visibly disruptedas a result of the treatment. The process was rapid, reproducible, andrequired less hands-on time than the control process. Typically, toobtain the same amount of material from the tissue as with theexperimental process, without the 5 use of sonic processing, wouldrequire three or more repeated methanol extractions using the controlprocess. As the sample is often at room temperature during this process,labile cellular constituents may be degraded. TABLE 2 Sample data forvarious treatments. Amino acids extracted in the first extraction as aSample Number percent of the total amount (randomly assigned) TreatmentDescription of extracted amino acids  4 Control process 86.5 10 Controlprocess 75.9 34 Control process 82.7 11 Sham control 83.1 22 Shamcontrol 92.4 31 Sham control 78.2  9 Mix, type one 90.7 49 Mix, type one96.8 16 Mix/dither, type two 99.2 46 Mix/dither, type two 99.9

[0243] TABLE 3 Averaged results of Table 2. Average amino acidsextracted in the first extraction as a percent of the total amount ofextracted amino acids (an average of the results for each treatment asCoefficient Treatment shown in Table 2) of variation Control process (3samples) 81.7% 6.6% Sham control (3 samples) 84.6% 8.5% Mix, type one (2samples) 93.8% 4.6% Mix/dither, type two (2 samples) 99.6% 0.5%

Example 6 Controlled Sample Mixing with Control of Both Cooling andHeating

[0244] This example illustrates ultrasound-forced convective cooling ofliquid samples. The experimental apparatus used in this example wassimilar to that used in Example 5, above. The waveform input to thetransducer consisted of 10,000 1.1 MHz frequency bursts with 1000 cyclesper burst and a 10% duty cycle. The amplitude was 500 mV (into a 55 dBRF amplifier). The cumulative dose was 10 million cycles. This waveformwas generated by LabVIEW software driving two function generatorsoperating in series. This is the “mix-and-treat” waveform used inExample 5, above. The sample vessel was a polypropylene vial with theends replaced with polyethylene film and held in the focal zone by afixture. The solvents used were either water or a 90% methanol solution.The sample vessel was filled to minimize headspace. The temperature wasmonitored with a ColePalmer, Model 39670-00 infrared sensor with a spotsize of 0.17″ (4.3 mm) at 0.0 inches distance and 1.0″ (2.5 cm) at 5inches (12.7 cm) distance. The sensor was connected to an Omega ModelDP116-JC2 temperature display. The sensor, with a response time of lessthan 450 milliseconds, was in close proximity with the sample top.

[0245] Treatment vessels were placed into a water bath at 3.5° C. Beforetreatment, the samples were allowed to equilibrate at a temperatureintermediate to the bath temperature and the ambient air temperature.Upon initiation of ultrasound treatment, the temperature of the samplesdropped from their initial temperature to a temperature near that of thewater bath and then rose above the initial temperature. This observationindicates that for the first few seconds of the treatment, a net coolingeffect was achieved by forcing convective heat transfer with ultrasonicenergy. The data are shown in Table 4, below. In the case of themethanol solution, the temperature was depressed below the startingequilibrium position for the first 40 seconds. In the case of the water,the temperature was depressed for the first 10 seconds. TABLE 4 Sampletemperature during treatment (degrees Celsius) as a function of thefluid in the sample treatment vessel and treatment time. Bathtemperature, in which the treatment vessels sit, is 3.5 degrees Celsius.Temperature of Treatment sample in degrees Temperature of sample in Time(seconds) Celsius (90% methanol) degrees Celsius (water) 0 6.5 6.5 5 4.64.5 10 3.8 5.3 15 3.9 6.8 20 4.4 7.8 25 5.0 8.2 30 5.6 8.4 35 5.9 8.5 406.4 9.1 45 7.2 9.8 50 7.4 9.8 55 7.8 10.0 60 8.5 10.2 65 9.4 10.5

[0246] These samples were treated with a powerful treatment waveformthat was developed to disrupt tissue samples. The waveform likely can bemodified to preferentially enhance the cooling effect such that thesample temperature would be depressed below the equilibrium temperaturefor as long as necessary. Other waveforms can be optimized for heatingand for treating the sample. In this way, the sample could besequentially heated, cooled, and/or treated. Both the heating andcooling waveforms promote mixing. This allows acceleration and controlof reactions that would otherwise be rate-limited by diffusion. Forexample, an enzymatic reaction occurring slowly in a cold solution, suchas one at 4° C., may be activated by application of a heating waveform.Following cessation of the heating waveform, the sample is rapidlychilled by a cooling waveform to inhibit the reaction. This rapidtemperature cycling is useful for thermal-cycle based protocols such aspolymerase chain reaction (PCR).

Example 7 Passive Cavitation Detection (PCD) to Monitor Efficiency ofUltrasonic Dosage

[0247] An apparatus can be assembled to measure cavitation induced by anultrasonic wave. One possible configuration uses a Panametrics A315R-SU,10 MHz transducer, optionally a Kron-hite 23 band-pass filter, aPanametrics 5676, 20 MHz, 40 dB pre-amplifier, a Panametrics 5607 gatedpeak detector, and a National Instruments, PCI-6111E, 5 MHz, two channelanalog acquisition card digitizer board. LabVIEW instrument controlsoftware is configured to analyze the signal produced by the gated peakdetector. This is considered “passive” cavitation detection, because itdetects acoustic signals generated directly by the motion and collapseof cavitation bubbles. Other useful active cavitation detection systemsare based on the scattering or modulation of laser light by cavitationbubbles.

[0248] Cavitation bubble collapse generates wide-band noise. Bubbles arevery effective scatterers of ultrasound. The pulsation mode of a bubbleis referred to as monopole source which is a very effective acousticsource. For small, generally linear oscillations, the bubble simplyscatters the incident acoustic pulse. However, as the response becomesmore nonlinear, it starts also to emit signals at higher harmonics. Whendriven harder the bubbles start to generate subharmonics as well.Eventually, as the response becomes a periodic or chaotic, the scatteredfield tends towards white noise. In the scenario where inertialcollapses occur, a short acoustic pressure pulse is emitted. An acoustictransducer can be configured to detect these emissions. There is astrong correlation between the onset of the emissions and cell lysis.

[0249] The PCD is normally arranged to be confocal with a high powertransducer so that it collects cavitation signals from the focus of thehigh power beam. The signal from the PCD is amplified and passed througha 2 MHz high-pass filter. The high-pass filter removes the 1 MHz signaldue to scattering of the fimdamental pulse and any other scatterers. Theamount of cavitation that the sample has been subjected to can beestimated by integrating the noise signal received by the PCD.

[0250] The signal generated by the cavitation detection system can beused as a feedback control element in an automated system. The automatedsystem controls the cavitation by either manipulating the sampleposition by dithering or other motion to affect the position ofcavitation nucleation sites, modulating or controlling the ultrasoundsignal, modulating or controlling overpressure (as in Example 8), and/orcontrolling the composition of dissolved gasses in the treatment vessel.

Example 8 The Application of Overpressure to Limit or Control Cavitation

[0251] A treatment vessel was overfilled with fluid prior to sealing thevessel. The interior fluid chamber was at a slight overpressure relativeto atmospheric pressure and the water bath pressure. This overpressureinhibited cavitation effects, such as tissue disruption within thesample vessel. When a sample of leaf tissue was placed in this settingand treated with the previously described experimental apparatus with awaveform consisting of 500 mV amplitude, 500 cycles/burst, 1,000 bursts,and 10% duty cycle input to a 55 dB amplifier, there was only a slighttissue disruptive effect. When the voltage was increased to 700 mV withthe same dosage parameters, there was no marked change in tissuedisruption.

[0252] When the sample vessel was opened to relieve the overpressure andthe sample was given a 500 mV dose, the tissue was disrupted. Theoverpressure apparently inhibited the cavitation bubble formation andcollapse that is related to and can cause tissue disruption. This resultdemonstrates that overpressure may be used to control or limitcavitation. Overpressure can be effectively integrated with apredetermined pattern of sonic energy exposure, both by altering thesonic energy and the location of the sonic energy relative to the sonicenergy focal zone, such that the disruptive effects of sonic energyexposure due to cavitation can be selectively muted with pressurecontrol. Additionally, in conjunction with a cavitation sensor thatprovides information to a control system through a feedback controlloop, controlled overpressure could be used to treat biological or othermaterials where it is desirable to control the intensity of thecavitation, such as in the controlled disruption or permeabilization ofbiological membranes.

Example 9 Treatment Vessel Design-Shape, Wall Thickness, and MaterialChoice

[0253] Several factors can be relevant in the design of a treatmentvessel. The treatment vessel geometric design shown in FIG. 5A depicts adevice with a dome shape that is able to transfer the heat generatedfrom the ultrasound process from the sample mixture to the surroundingwater bath. Example 2, above illustrates the use of this dome-shapeddevice and the importance of good heat transfer and acoustictransparency characteristics. Typically, the material from which atreatment vessel is constructed should absorb relatively little sonicacoustic energy and should impede sonic energy at a level that issimilar to the sonic energy impedance of water. The thickness of thematerial also should be relatively thin, for maximizing ultrasoundtransmission, maximizing heat transfer between the interior and exteriorof the vessel, and facilitating monitoring of the treatment vessel andit contents, by, for example, infra-red temperature sensors, cavitationdetection sensors, and/or video or optical monitors.

[0254] Standard polystyrene or polypropylene microwell plates, such as96 well plates, have wall and bottom thickness of approximately 1 mm.Tests with a microwell plate, oriented in a horizontal plane, that isexposed to sonic energy from a needle-tip transducer hydrophone with theacoustic path completely submerged, resulted in approximately 70%transmission through polystyrene. The resultant absorption issignificant in a high power dosage. For example, a 1.1 MHz continuouswave sample dosage of 500 mV input to a 55 dB RF amplifier for 30seconds applied to a polypropylene microwell plate generates enough heatto bring 300 microliters of ice to a boil within seconds.

[0255] Temperature rise was measured in two different vessels, the“bubble-wrap” vessel described in Example 2 and a polypropylene vial,using a mix-treat waveform of 10% duty cycle, 10,000 bursts, 1,000cycles/burst at 500 mV as described in the examples above. Variousvolume/volume ratios of methanol in water were evaluated, including 0%methanol, 50% methanol, 90% methanol, and 100% methanol. In each case,the starting temperature was close to 1° C. The results shown in Table 5are the magnitudes of temperature rise following dosage. The temperaturewas measured with the infrared sensor and meter, as described in theexamples above. TABLE 5 Temperature rise in degrees Celsius as afunction of methanol concentration and sample vessel type. Temperaturerise of methanol Temperature rise Methanol solution in of methanolsolution (volume “bubble-wrap solution in methanol/ vessel”polypropylene vial volume water) (degrees Celsius) (degrees Celsius) 0%0.0 4.4 50% 2.2 6.3 90% 5.4 7.2 100% Not determined 10.5

[0256] These results show that the bubble-wrap vessel of Example 2 isbetter suited to samples that should remain substantially isothermalduring treatment than is a polypropylene vial.

Example 10 Sonic Energy Optimization

[0257] A series of experiments was performed using the apparatusdescribed above to optimize exposure of a sample to sonic energy. First,a wavetrain was optimized for both treatment and mixing. Briefly, and asmore fully described in Example 2, a sample treatment vessel wasconstructed from a ⅜ inch (9.5 mm) diameter hemispherical “bubble” takenfrom bubble-wrap packaging material. The flat side of the bubble was cutopen with a hot-knife to access the interior. The sample treatmentvessel was held in a metal fixture such that the focal zone of theacoustic field was within the vessel. The sample treatment vessel had avolume of approximately 300 microliters.

[0258]A. thaliana leaf tissue was prepared by freezing, lyophilizing,and grinding. Approximately 25 milligrams (dry weight) of tissue was putinto the sample treatment vessel. This tissue was rehydrated with 200microliters of a 50% MeOH solution. A layer of plastic saran film 0.001inches (0.025 mm) thick was positioned on the flat side of the treatmentvessel and the whole was clamped in a metal fixture.

[0259] Several different acoustic wavetrains were applied to the sample.The mixing effect was judged visually. The temperature rise was measuredwith a J-type thermocouple at the periphery of the treatment vesselusing an Omega DP116-JC2 meter. The parameters being varied include thenumber of cycles per burst and the duty cycle. Fixed parameters includedthe 1.1 MHz frequency of the cycles and the 500 mV amplitude applied toa 55 dB RF amplifier and thereafter to the transducer. The results ofvarying the parameters are shown in Table 6, below. TABLE 6 Results ofvarying the number of cycles per burst and the duty cycle on heating ofthe sample and mixing of the sample. Temperature Rise Cycles per ofSample Mixing Effect Burst Duty Cycle in degrees Celsius in Sample 5 1%1.5 none 500 10% 3.5 none 10,000 20% 6.0 slight stirring 10,000 10% 4.0some stirring 1,000 10% 3.2 good mixing

[0260] Repeated trials of the final combination, 1,000 cycles per burstand 10% duty cycle, showed that this combination is effective at mixingthe sample material in the sample treatment vessel. This combination ofparameters produces a compromise wavetrain that both mixes and treatsthe sample.

[0261] Further experimentation verified the ability to separate theheating and mixing functions, such that each function can be optimizedindependently of the other and apparatus and methods of the presentinvention can alternate between these two functions. More particularly,a treatment wavetrain was alternated with a mixing wavetrain. In thisseries of experiments, the sample was approximately 200 micrograms offresh-frozen A. Thaliana in 600 microliters of water. The sampletreatment vessel was a polypropylene cylinder with an inside diameter of0.5 inches (1.3 cm) and a length of 1.7 inches (4.3 cm). The open end ofthis cylinder was covered with 0.001 inch (0.025 mm) thick polyethylenefilm as an acoustic window. The treatment vessel was positioned suchthat the focal zone of the sonic energy was inside the treatment vesseland the majority of the acoustic energy passed through the polyethylenefilm window.

[0262] The treatment wavetrain was 5 cycles per burst, a 1% duty cycle,500,000 total cycles, and a 500 mV amplitude into the RF amplifier andthereafter to the transducer. The mixing wavetrain consisted of CWultrasound at 100 mV for 500,000 cycles. The treatment wavetrain wasfollowed by the mixing wavetrain. Each wavetrain takes about 0.5 secondsto complete. The treatment wavetrain followed by the mixing wavetrainwas repeated 5 times. Mixing was determined by examining the sampleduring treatment to visualize tissue particles moving in the treatmentvessel. Treatment was determined by examining the tissue samples under astereo microscope for the creation of small tissue particles and/or theshredding of larger tissue particles.

[0263] The mixing wavetrain was effective and good mixing of the samplewas visually apparent. The treatment wavetrain was partially effective;the smaller fragments of tissue were treated but the larger ones werenot substantially treated. This experiment demonstrates that a treatmentwavetrain can be alternated with a mixing wavetrain to achieve bothtreatment and mixing. Alternating treatment and mixing wavetrains allowseach to be optimized for its specific function.

Example 11 Sonolysis of Plant Leaf Tissue

[0264] A 70 mm diameter focused ceramic piezoelectric transducer domewas inserted into a water tank and the focal point domain was defined tobe approximately 62 cm away from the surface of the dome. A continuous,sinusoidal wave form of 1 MHz frequency with 0.2 Volt from thepreamplifier with a resultant 5 MPa positive pressure from the focusedpiezoelectric transducer was generated for short time durations of 1 and10 seconds. The focused energy zone was approximately 3 mm diameter by 6mm in length. Approximately 100 mg of Arabidopsis thaliana leaf tissuehad been collected and immediately frozen in liquid nitrogen and storedat −70° C. until use. Tissue samples were inserted into 0.325 ml of CTABbuffer (1M TRIS pH 7.5, 200 ml, CTAB (Hexadecyltrimethyl AmmoniumBromide) 20 g, NaCl 81.76 g, 0.5 M EDTA pH 7.5 40 ml, H₂O 1,500 ml) intoa standard flat-bottom, polystyrene 96-well microtiter plate (Immulon1B, cat. 3355, Dynex Technologies, Chantilly Va.). In some otherprotocols, 10 μl 2-mercaptoethanol/ml CTAB buffer is added prior to use.For this experiment it was omitted.

[0265] After tissue and buffer were applied, a 0.010″ thick (0.25 mm)film of acetate sealing tape for microtiter plates (catalog no.001-010-3501, Dynatech Laboratories, Chantilly, Va.) with adhesive wasapplied to cover the samples. The plate was kept on dry ice until use.The plate was loaded onto a x, y, z positioning fixture that wascontrolled by LabView software. The plate was lowered into the immersiontank filled with deionized water at room temperature, approximately 22°C. The plate was positioned so that one well was in the focus of theceramic piezoelectric dome. The dose was applied through the film andnot through the bottom of the well. The duration and amplitude, werevaried for the exposure period.

[0266] Analysis of material following exposure revealed the exposed leaftissue sample supernatants were green, whereas control samplesupernatants were only slightly green, likely due to leaching ofchlorophyll from the cut ends of the tissue. The treated tissue, whenviewed under a dissecting microscope, appeared thinner, moretranslucent, and with small “bubbles” appearing below the initialsurface layers. Samples that had been exposed in the presence of glassbeads (Sigma, 212-300 microns, unwashed G-9143, lot No. 75H0617) werenot affected as much, based on the observation that both the buffersolution was clearer and the microscopic structure of the leaf tissuewas different. The leaf tissue appearance was thicker and filled withlarger and more “bubbles” below the external surface layers. The glassbeads may have absorbed or reflected some of the energy applied to thesample. In addition, if the samples were oriented with the bottom of thepolystyrene plate between the dome and the sample, the samples were notnoticeably affected, however, with 0.5 V for 20 seconds, melting of thepolystyrene bottom occurred. The polystyrene material may have absorbedthe energy in a continuous wave duration of 20 seconds.

Example 12 Focused Sonolysis with Automated Extraction

[0267] Biological material is inserted into a microtube system, wherethe bottom of the tube is a semi-permeable material such as hydrophobicmembrane. Under atmospheric pressure, the membrane contains bulk waterand under negative pressure allows liquid water and cellularconstituents to traverse. Ideally, the material is transparent toacoustic energy, or at least having similar acoustic properties as thefluid through which the sound energy is transmitted. A leaf tissuesample is placed into a tube with 0.35 ml of CTAB buffer, as in Example1, and frozen. The frozen sample is placed within the focus of a domedultrasonic transducer. The sample is exposed to sonic energy generatedby a 0.5 V signal input to a 55 dB RF amplifier at 1 MHz for 2 seconds.The sample is removed from the exposure chamber and allowed to thaw to4° C. The sample is then vortexed for 10 seconds, and the filtrate isremoved by inserting the tube into a holder and centrifuging the samplefor 10 minutes at 10,000×g. A wash of 0.5 ml of CTAB buffer is appliedto the previously spun sample, the leaf tissue is resuspended in thebuffer, vortexed, and spun as described above. The extract is analyzedfor DNA content.

Example 13 Focused Sound Waves on Frozen Tissue with Real-TimeTemperature Control

[0268] 100 μg of leaf tissue is frozen in liquid nitrogen. The frozenmaterial is added directly to a microtube, as described in Example 2,and is suspended in a bath of ethylene glycol chilled to about −15° C.Immediately, 0.25 ml of buffer, chilled to approximately 4° C., isaliquoted into the tube with the tissue. The sample buffer is chilled tobelow 0°. The microtube has been aligned in the focal point of thepiezoelectric transducer. As the sample is chilling, focused sound wavesare applied to the sample. The wavelength, duration, and amplitude aremodulated a priori on test samples to approximate total energy appliedto the system. The system can also utilize a closed-loop feedbackmechanism with an external temperature probe to monitor temperature risein the sample. The sample should be treated with sound waves to inducedisruption, but the temperature should preferably not be elevated aboveabout 0° C.

Example 14 Acoustic Transmission Properties of Polymeric Materials

[0269] The acoustic transparency of materials may be measured in orderto design optimal and reproducible shock treatment protocols. To testthe acoustic transparency of various polymeric materials, a submergedultrasonic transducer in a water bath was focused on a submergedmicrotiter plate of the type which had wells open at the bottom. Anultrasonic needle-tip hydrophone was placed behind the plate to measurethe transmission across the microtiter plate. The bottom of the platewas blocked with various materials of possible use in the proposedextraction techniques. The transducer was set into a continuous wavegeneration module at low voltage. As shown by the data in Table 7,below, polyethylene terephthalate (PET) material caused the leastattenuation of the acoustic intensity, of these materials. Equivalent orbetter performing materials can be readily identified using routineexperimentation in accordance herewith. TABLE 7 Relative sonic energytransmission through various materials Relative transmission of sonicenergy (0.05 V Thickness at 1 MHz for Material of material 10 seconds)No plate 100% Acetate 0.005 inch 80% Latex 0.004 inch 50% PET (Mylar)0.005 inch 90% Silicone 0.005 inch 95% PET (Mylar) 0.002 inch >95%

Example 15 High Intensity Focused Ultrasound Disruption of Leaf Tissue

[0270]Arabidopsis thaliana leaf was collected and immediately immersedin liquid nitrogen. Samples were then stored in a −80° C. freezer untiluse. Leaf samples were inserted into prechilled microtiter plates filledwith approximately 300 ml of refrigerated, precooled CTAB lysis bufferas described in Example 1.

[0271] The leaf sample stalk was removed with a single-edge razor bladeon a precooled surface such as a dry ice chilled cutting block. Theremaining frozen tissue was inserted into the microwell and the leaf inthe lysis buffer was either frozen or kept at 4-8° C. until use. Themicrowell plate was affixed to an x, y, z positioning system toautomatically align the samples prior to dosage. The sample plate waspreviously aligned in an insulated bath vessel filled with ethyleneglycol that had an acoustically transparent window on the bottom. Theenergy system enables a transducer submerged in a water bath below thesample bath vessel to transmit a focused sound wave through the aqueoustransducer bath, then through the acoustic window into the sample bath,and through the ethylene glycol liquid in the sample bath to the bottomof the microwell.

[0272] The pulse then entered the CTAB lysis buffer inside the samplewell, and was focused on the leaf tissue. In this example, the focus ofthe system was aligned by applying a low voltage ultrasonic CW and bymonitoring bubbling on the surface. The peak focal point was tested tobe 2 mm×4 mm and measured with a commercially available needle-pointhydrophone that fit into the microwells.

[0273] Using a suitable submersible transducer, energy from the sourcewas applied as a continuous wave, generated over approximately 1 MPapositive peak pressure and approximately −1 MPa of negative peakpressure at 50 mV modulation amplitude and at 1 MHz frequency. At thisvoltage, the waveform approximated a symmetrical harmonic. However, asthe voltage increased, the peak positive pressure increased, while thepeak negative pressure became less pronounced. For example, at 700 mV at1 MHz, the peak positive pressure was over 22 MPa (−3,100 psi) and thepeak negative pressure was only −9 MPa (˜−1,300 psi). The repetitivecomplex waveform had sharp positive pressure peaks and blunted negativepressure peaks, due to the nonlinear behavior of the fluid medium(water). The negative pressures are thought to contribute to cavitation.

[0274] Using the above apparatus and tuning, three sets of variabledoses were given to the leaf tissue in microtiter plates. In Series A,pulses were applied continuously for 2 million cycles over about a 2seconds period. At 50 mV, there was no discernible effect on the sample.At 100 and 200 mV there was also no effect, but at 500 mV amplitude, thesample was full of bubbles and froth, and the extraction buffer turnedgreen with extracted material. When the amplitude was raised to 700 mV,the bottom of the polystyrene plate began to melt. Thus, a certain rangeof energy intensity is effective in Series A.

[0275] In Series B one burst, which was three cycles long, was appliedto the samples. No extraction of material from the leaf disc wasobserved at any of the voltage levels used, including 700 mV. Thus, aminimum amount of energy is required. Three cycles at these energylevels does not appear to be enough.

[0276] In Series C, a 3 cycle burst was applied 100 times. There was noeffect at 50, 100 and 200 mV. At 500 mV the solution became slightlygreen, indicating the beginning of extraction. At 700 mV, the solutionbecame definitely green, indicating substantially complete extraction.There was no severe bubbling, or any melting. Thus, it isstraightforward to determine appropriate operating conditions for theuse of the extraction system of the invention on a particular materialin a particular arrangement.

Example 16 Temperature Effects

[0277] Using the apparatus of Examples 14 and 15, another series ofexperiments compared a slightly above-freezing extraction temperature(6° C. +/−2° C.) versus a slightly below freezing temperature (−4° C.+/−1° C.). For all experiments, the doses were 100 mV, 200 mV, 500 mV,and 0 mV (control) with the 0, 200, and 500 doses in duplicate. Thenumber of bursts was varied. The condition of the tissue was observed inthis experiment, in contrast to the appearance of the extraction buffer,as in the previous example.

[0278] Series A—Above Zero Temperature

[0279] 500 bursts—The control leaf tissue samples (0 mV; no powerapplied) were bright green with full appearance. There was no markeddifference in the experimental samples except that they were slightlymore transparent. Near the tip, closest to the sample source, thereappeared to be bubbles connected in strands.

[0280] 1,000 bursts—the control was bright green with full tissueappearance. No marked difference was seen among experimental samples.Again, there was a slight appearance of bubble channels with slightlymore at tips of leaves.

[0281] 5,000 bursts—only the 100 mV experimental appeared close to thecontrol. All of the other samples had micro channels of linked bubbles.The samples that had the highest dose had fewer microchannels thansamples receiving lower doses at 5,000 bursts, indicating that acousticinterference with bubble formation can be a consideration when choosingconditions for disrupting a particular sample.

[0282] Series B—Below Zero Temperature

[0283] 500 bursts—Control was intact tissue with no sign ofmicrochannels. The low dose of 100 mV had a few independent bubbles, andthe 200 mV sample had many independent bubbles (no microchannels oflinked bubbles). Samples in the 500 mV wells also had independent,discrete bubbles.

[0284] 1,000 bursts—control and 500 mV had slight bubbles. All othersamples had more discrete, independent bubbles.

[0285] 5,000 bursts—the control had slight bubbles formed, but not asmany bubbles as with the 1,000 burst series. The remaining tissueappeared thin, transparent, and had an apparent loss of cell structure.The bubbles in the control sample likely indicated spillover of energyfrom the adjacent well, which also was exposed to the most energy. Thisobservation suggests that a microtiter plate can be made of a materialsuch that the walls of the wells are capable of absorbing acousticenergy.

[0286] Following three days storage between two glass microscope slidesat room temperature with isolated leaf tissue prepared at subzerotemperatures, all of the control tissue appeared green, while theexperimental tissues that had 100 and 200 mV doses appeared virtuallytransparent, especially the 100 mV samples. The results indicate thatsubstantial disruption of the plant leaf tissue occurred as a result ofthe dosage when the process was performed at sub-zero temperatures andthe pulse duty cycle minimized sample heating.

[0287] Features, specifications, and functionality of the hardware,operating software, sonic energy profile, and positioning profile ofcertain embodiments of a system according to the invention are describedin FIGS. 10-13. As noted, some of these embodiments can be usedeffectively for treating a sample for the purpose of extraction ortransformation or general research; however, these embodiments are to beconsidered exemplary in nature, and not limiting of the invention.

[0288] While there has been described herein what are considered to beexemplary and preferred embodiments of the invention, othermodifications and alternatives of the inventions will be apparent tothose skilled in the art from the teachings herein. All suchmodifications and alternatives are considered to be within the scope ofthe invention.

[0289] Accordingly, what is desired to be secured by Letters Patent isthe invention as defined and differentiated in the following claims andequivalents thereof.

What is claimed is:
 1. An apparatus for processing a sample using sonicenergy, the apparatus comprising: a sonic energy source for emittingsonic energy; a holder for the sample, the sample movable relative tothe emitted sonic energy; and a processor for controlling the sonicenergy source and location of the sample according to a predeterminedmethodology, such that the sample is selectively exposed to sonic energyto produce a desired result.
 2. The apparatus of claim 1 furthercomprising a feedback system connected to the processor for monitoringat least one condition to which the sample is subjected duringprocessing, such that the processor controls at least one of the sonicenergy source and the location of the sample in response to the at leastone condition.
 3. The apparatus of claim 1 wherein the desired result isselected from the group consisting of heating the sample, cooling thesample, fluidizing the sample, mixing the sample, stirring the sample,disrupting the sample, increasing permeability of a component of thesample, enhancing a reaction within the sample, and sterilizing thesample.
 4. The apparatus of claim 1 further comprising a temperaturecontrol unit for controlling temperature of the sample.
 5. The apparatusof claim 4 wherein the processor controls the temperature control unit.6. The apparatus of claim 1 further comprising a pressure control unitfor controlling pressure to which the sample is exposed.
 7. Theapparatus of claim 6 wherein the processor controls the pressure controlunit.
 8. The apparatus of claim 1 wherein the sonic energy sourcecomprises a transducer.
 9. The apparatus of claim 8 wherein thetransducer focuses the sonic energy.
 10. The apparatus of claim 8wherein the transducer is selected from the group consisting of at leastone piezoelectric element, an array of piezoelectric elements, anelectrohydraulic element, a magnetostrictive element, an electromagnetictransducer, a chemical explosive element, a laser-activated element, andcombinations thereof.
 11. The apparatus of claim 10 wherein the at leastone piezoelectric element includes a spherical transmitting surfaceoriented such that the focal axis is vertical.
 12. The apparatus ofclaim 1 wherein the holder supports a sample container for containingthe sample.
 13. The apparatus of claim 12 wherein the sample containeris selected from the group consisting of a membrane pouch, athermopolymer well, a polymeric pouch, a hydrophobic membrane, amicrotiter plate, a microtiter well, a test tube, a centrifuge tube, amicrofuge tube, an ampoule, a capsule, a bottle, a beaker, a flask, anda capillary tube.
 14. The apparatus of claim 12 wherein the samplecontainer forms multiple compartments.
 15. The apparatus of claim 12wherein the sample container includes a rupturable membrane fortransferring a fraction of the sample away from the holder.
 16. Theapparatus of claim 1 further comprising a device for moving the samplefrom a first location to a second location.
 17. The apparatus of claim16 wherein the device for moving the sample comprises a stepper motor.18. The apparatus of claim 2 wherein the feedback system comprises asensor for monitoring the at least one condition.
 19. The apparatus ofclaim 1 further comprising an acoustically transparent material disposedbetween the sonic energy source and the holder.
 20. The apparatus ofclaim 1 wherein the desired result comprises an in vitro treatment. 21.The apparatus of claim 1 wherein the desired result comprises an ex vivotreatment.
 22. The apparatus of claim 1 wherein the sample flows througha conduit.
 23. The apparatus of claim 1 wherein the sonic energy sourcegenerates sonic energy at two different frequencies.
 24. The apparatusof claim 1 wherein sonic energy source generates a wavetrain.
 25. Theapparatus of claim 24 wherein the wavetrain comprises a first wave and adifferent second wave.
 26. The apparatus of claim 24 wherein thewavetrain comprises about 1000 cycles per burst at about a 10% dutycycle at about 500 mV.
 27. A method for processing a sample with sonicenergy, the method comprising the steps of: exposing the sample to sonicenergy; and controlling at least one of the sonic energy and location ofthe sample relative to the sonic energy according to a predeterminedmethodology, such that the sample is selectively exposed to sonic energyto produce a desired result.
 28. The method of claim 27 furthercomprising the steps of sensing at least one condition to which thesample is subjected during processing and altering at least one of thesonic energy and the location of the sample in response to the at leastone condition.
 29. The method of claim 28 wherein during the sensingstep, the at least one condition is selected from the group consistingof temperature, pressure, an optical property, an altered chemical, anacoustic signal, and a mechanical occurrence.
 30. The method of claim 28wherein during the altering step, at least one characteristic of thesonic energy is altered, the at least one characteristic selected fromthe group consisting of wave form, duration of application, intensity,and duty cycle.
 31. The method of claim 27 wherein the desired result isselected from the group consisting of heating the sample, cooling thesample, fluidizing the sample, mixing the sample, stirring the sample,disrupting the sample, increasing permeability of a component of thesample, enhancing a reaction within the sample sterilizing the sample,and combinations thereof.
 32. The method of claim 27 further comprisingthe step of controlling temperature of the sample.
 33. The method ofclaim 27 further comprising the step of controlling pressure to whichthe sample is exposed.
 34. The method of claim 27 wherein during thestep of exposing the sample to sonic energy, the sonic energy isgenerated by at least one process selected from the group consisting ofspark discharges across a gap, laser pulses, piezoelectric pulses,electromagnetic shock waves, electrohydraulic shock waves, electricaldischarges into a liquid, and chemical explosives.
 35. The method ofclaim 27 wherein the sonic energy is focused on the sample.
 36. Themethod of claim 27 wherein the sample contains a cell, the methodfurther comprising the step of introducing a material into the cell. 37.The method of claim 36 wherein the material is selected from the groupconsisting of a polymer, an amino acid monomer, an amino acid chain, aprotein, an enzyme, a nucleic acid monomer, a nucleic acid chain, asaccharide, a polysaccharide, an organic molecule, an inorganicmolecule, a vector, a plasmid, a virus, and combinations thereof. 38.The method of claim 27 further comprising the step of extracting acomponent of the sample.
 39. The method of claim 27 wherein during thecontrolling step, at least one characteristic of the sonic energy iscontrolled, the at least one characteristic selected from the groupconsisting of wave form, duration of application, intensity, and dutycycle.
 40. The method of claim 27 wherein the desired result comprisesan in vitro treatment.
 41. The method of claim 27 wherein the desiredresult comprises an ex vivo treatment.
 42. The method of claim 27further comprising the step of the sample flowing through a conduit. 43.The method of claim 27 wherein the sonic energy comprises at least twodifferent frequencies.
 44. The method of claim 27 wherein sonic energysource comprises a wavetrain.
 45. The method of claim 44 wherein thewavetrain comprises a first wave and a different second wave.
 46. Themethod of claim 44 wherein the wavetrain comprises about 1000 cycles perburst at about a 10% duty cycle at about 500 mV.